Compositions for conferring immunogenicity to a substance and uses thereof

ABSTRACT

A multimer of monomers non-covalently held together by interactive peptide linkers is provided for the enhancement of the immunogenicity of a substance. These multimers are useful for stimulating or suppressing the immune system, detecting the presence of antibodies, bypassing MHC restriction in an animal, the effective presentation of antigen, suppressing autoimmune disease, inducing cytokine production, adsorption, treating a defective immune system and for use as an adjuvant.

FIELD OF THE INVENTION

The present invention relates to the field of immunology and to theenhancement of the immunogenicity of a substance. A new composition ofmatter is provided, a multimer of monomers non-covalently held togetherby interactive peptide linkers. These multimers are useful forstimulating or suppressing the immune system, detecting the presence ofantibodies, bypassing MHC restriction in an animal, effectivepresentation of antigen, suppressing autoimmune disease, inducingcytokine production, adsorption, treating a defective immune system andfor use as an adjuvant.

BACKGROUND OF THE INVENTION

Immunogenicity refers to the ability of a substance to induce adetectable immune response (humoral and/or cellular) when introducedinto an animal. Such substances are called "immunogens." Antigenicityrefers to the ability of a substance to bind to an antibody that isinduced by exposure to an immunogen. Such substances are calledantigens. Substances having a low molecular weight, such as drugs andantibiotics, are non-immunogenic, however, when coupled to animmunogenic protein, will induce antibodies having specificity for thelow molecular weight substance. The terms "epitope" and "antigenicdeterminant" refer to the part of the antigen that is bound by antibodyor T cell receptor.

Proteins are the most potent immunogens, but polysaccharides, syntheticpolypeptides, and other synthetic polymers such as polyvinylpyrrolidoneare immunogenic under certain conditions. For a general discussion ofimmunogenicity, a chapter authored by Goodman (1991) is incorporated byreference herein.

Immunogenicity is not an inherent property of a molecule but isdependent on experimental conditions. These conditions include theparticular immunogen, the route of immunization, the host beingimmunized, and the sensitivity of the detection methods. Factors thatconfer immunogenicity on molecules are complex and incompletelyunderstood, but certain conditions must be satisfied in order for amolecule to be immunogenic (Goodman, 1991). Those conditions include"foreignness", molecular size, chemical complexity, the geneticconstitution of the animal, and the method of administration.

Foreignness

The immune system normally discriminates between "self" and "nonself,"so that only molecules that are foreign to the animal are immunogenic.

Molecular Size

Small molecules such as amino acids or monosaccharides are not usuallyimmunogenic, and it is generally accepted that a certain minimum size isnecessary for immunogenicity. However, there is no specific sizethreshold below which all substances are inert and above which all areactive. In a few instances, substances with molecular weights of lessthan 1000 have proved to be immunogenic, but as a general rule,molecules smaller than molecular weight 10,000 are only weaklyimmunogenic or not immunogenic at all. The most potent immunogens aremacromolecular proteins with molecular weights greater than 100,000.

Chemical Complexity

A molecule must possess a certain degree of chemical complexity to beimmunogenic. For example, synthetic polypeptides having repeating unitsof a single amino acid are poor immunogens regardless of size, whereascopolymers of 2 or 3 amino acids may be active. It is difficult toestablish a definite threshold, and the general rule is thatimmunogenicity increases with structural complexity. Aromatic aminoacids tend to contribute more to immunogenicity than non-aromaticresidues. For example, relatively simple random polypeptides containingtyrosine are better antigens than the same polymers without tyrosine.

Genetic Constitution of the Animal

The ability to respond to a particular immunogen is a function of theway the immune response is controlled genetically. It has been known forsome time that pure polysaccharides are immunogenic when injected intomice and humans but not when injected into guinea pigs. Further, strain2 guinea pigs respond readily in an easily detectable manner topoly-L-lysine, whereas strain 13 guinea pigs do not. The ability torespond is inherited as an autosomal dominant trait.

Method of Immunogen Administration

Whether an immunogen will induce an immune response depends on the doseand the mode of administration. A quantity of immunogen that isineffective when injected intravenously may evoke a copious antibodyresponse if injected subcutaneously in adjuvant. In general, once thethreshold is exceeded, increasing doses lead to increasing, but lessthan proportionate, responses. However, excessive doses may not onlyfail to stimulate antibody formation; they can also establish a state ofspecific unresponsiveness or tolerance.

Immunogenicity can be enhanced if the immunogen is mixed with substancestermed adjuvants. Adjuvants function as follows: (1) by prolongingretention of the immunogen; (2) by increasing effective immunogen size,(3) by stimulating the influx of populations of macrophages and/orlymphocytes, or (4) by inducing certain cytokines. The most potentadjuvant for use in experimental animals is Freund's complete adjuvant(FCA), a water-in-oil emulsion containing killed mycobacteria. Theadjuvant effect of FCA is aided by the provision of a depot for theimmunogen and stimulation of macrophages and certain lymphocytes, butits very strong inflammatory effect precludes its use in humans. Amuramyl dipeptide constituent of mycobacterial cell walls has also beenfound to possess adjuvant activity. The most widely used adjuvant inhumans is a suspension of aluminum hydroxide on which the immunogen isadsorbed (alum precipitate). This adjuvant increases the effectiveparticle size of the immunogen, promoting its presentation tolymphocytes.

When examined empirically, the amino acid residue sequences of peptidesknown to bind to major histocompatibility complex (MHC) binding sites,(Guillet et al., 1987; Sette et al., 1989; Hale et al., 1989), have astriking consistency of pattern of alternating hydrophilic andhydrophobic amino acid sequences separated occasionally by more neutralamino acids. The precise roles of alternating hydrophobic andhydrophilic amino acids in this or any protein binding system areunknown.

One method of preparing higher molecular weight peptides for use invaccines has been published by Tam et al. (1988). In this method, amultimeric peptide antigen was chemically synthesized by covalentlybinding epitope subunits to amino groups of lysine cores. Eight tosixteen peptides could be incorporated into multimers using this method.The multimers were immunogenic in some mice but not others; and some,but not all, of the antibodies produced reacted with the native protein.There was a high probability that these multimers would denature. Infact, the aggregated forms were more useful in antibody ELISA assays.

Two promising new approaches to vaccine development have emerged in themodern era of biomedical technology. One is the cloning of genes codingfor important surface proteins of infectious agents, with production oflarge quantities of the desired protein by microorganisms transfectedwith the gene. A recombinant vaccine containing the major surfaceprotein of the hepatitis B virus has recently been approved and marketed(Goodman, 1991).

The other approach is the chemical synthesis of short peptides fromknown sequences of proteins from infectious organisms. The peptides maybe linked to carriers, thereby becoming "synthetic immunogens. " Thisapproach is predicated on the assumption that antibodies induced toshort peptides of the order of 6-15 amino acids will react with thehomologous sequences in the native proteins. In fact, it has been shownthat antibodies to peptides representing sequences from the exposedsurfaces of folded proteins, where they are accessible to antibody, doreact with the native molecules, although the affinities of binding maybe lower than with the peptides themselves. These findings offer promisefor the manufacture of synthetic vaccines that are based on thehapten-carrier principle for use in human and animal prophylaxis.However, an important consideration is that immunologic memory in theresponse to hapten-carrier conjugates is directed primarily at thecarrier, which bears the immunogenic determinants. Since the carriersare different in the synthetic vaccine and the native protein from whichthe peptide came, an encounter with the infectious agent followingimmunization with the synthetic vaccine should elicit little or nomemory against the peptide and its carrier. Although sufficiently highantibody titers raised by the vaccine could provide substantialprotection even without memory, this does represent a serious limitationfor this type of vaccine. Due to limitations such as these, those ofskill in this art have tried new approaches to enhancing theimmunogenicity of substances for the preparation of synthetic vaccines.

SUMMARY OF THE INVENTION

The present invention provides linkers having amino acid sequences ofalternating hydrophobic and hydrophilic amino acid(s) for attachment tothe amino and carboxy terminal ends of a peptide to form a monomer. Thelinkers bind strongly to each other in a non-covalent manner, thereby,multimerizing the monomer and conferring immunogenicity to themultimerized peptide.

The present invention provides the synthesis of protein molecules withrepeating active sites for any particular biological activity. Themultimers of the present invention are effective because: 1) thesubunits are not held together by covalent bonds but by natural aminoacid side chain interactions, thus allowing their tertiary conformationto be more flexible and natural; 2) they allow a greater number andvariety of immunogens, antigens, epitopes or ligands to be easilyincorporated into them; 3) since the linkers are not covalently bound,their flexibility allows a greater chance for the incorporatedimmunogen, antigen, epitope or ligand to have high affinity interactionwith its appropriate receptor; and 4) the binding of the monomers in thesystem of the present invention does not require the use of potentiallytoxic chemicals or a large amount of manipulation that can lead toprotein denaturation. The present non-covalently linked multimericpeptides are also sufficiently soluble to use in vitro or in vivo asimmunogen, antigen, epitope or receptor binders and/or stimulators. Inaddition, the present non-covalently linked multimeric peptides have thepotential of being produced by recombinant technology or used directlyas virally or, bacterially, expressed vaccines.

The present invention provides a non-covalently interlinked multimercomprising monomers (A)-Ag-(a) and (B)-Ag-(b). In this muiltimer, Ag ispreferably an HP-6 epitope but may be a peptide having from about 5 to30 amino acids. A, a, B, and b are independently peptide linkers ofabout 3 to 12 amino acids, where peptide linker A has binding affinityfor peptide linker b, and peptide linker a has binding affinity forpeptide linker B due to a pattern of alternating hydrophilic andhydrophobic amino acids or groups of amino acids. The multimer hereindefined preferably has immunogenicity.

In a preferred embodiment of the present invention, A and B are the samelinker and a and b are the same linker. The pattern of hydrophilic andhydrophobic amino acids may be one of singly alternating hydrophilic andhydrophobic amino acids or may be one of alternating groups of 2 or 3amino acids. The peptide linkers may have spacer amino acids.

In a particularly preferred embodiment of the present invention, A and aare selected from the group consisting of S1-L and S1-R, S2-L and S2-R,M1-L and M1-R, M2-L and M2-R, S20-L and S20-R, and S22-L and S22-R; andB and b are selected from the group consisting of S1-L and S1-R, S2-Land S2-R, M1-L and M1-R, M2-L and M2-R, S20-L and S20-R, and S22-L andS22-R.

The present invention also provides for a monomer comprising thestructure (A)-Ag-(a) wherein Ag is an HP-6 epitope; and A and a areindependently peptide linkers of about 3 to 12 amino acids. In thismonomer, peptide linker A has non-covalent binding affinity for peptidelinker a due to a pattern of alternating hydrophilic and hydrophobicamino acids or groups of amino acids.

A further embodiment of the present invention is a set of peptidelinkers for enhancing immunogenicity of a substance when covalentlylinked with the substance. The set of linkers comprises a first and asecond peptide linker, each linker having between about 3 to 12 aminoacids and having a sequence of alternating hydrophilic and hydrophobicamino acids or groups of amino acids. The first peptide linker hasnon-covalent binding affinity for the second peptide linker due to thesequence of alternating hydrophilic and hydrophobic amino acids orgroups of amino acids.

The present invention also provides a non-covalently interlinkedmultimer made by a process comprising the step of incubating a monomer,(A)-Ag-(a), with a monomer, (B)-Ag-(b), under conditions facilitatingpeptide interactions to form a non-covalently interlinked multimer. Inthis monomer, Ag is an HP-6 epitope; and A, a, B, and b areindependently peptide linkers of about 3 to 12 amino acids; whereinpeptide linker A has non-covalent binding affinity for peptide linker band peptide linker a has non-covalent binding affinity for peptidelinker B due to a pattern of alternating hydrophilic and hydrophobicamino acids or groups of amino acids. In this process, the conditionsfacilitate dissolution of the monomers in a physiologically acceptableaqueous solvent.

The term "peptide linker A having binding affinity for peptide linker b"means that upon contacting linker A with linker b, for example, underappropriate conditions of ionic strength, temperature, pH and the like,specific interaction will occur. The term "binding affinity" means thatthe linkers interact with an association constant of at least 10⁴. Theinteraction may occur due to specific electrostatic, hydrophobic,entropic or other interaction of alternating hydrophobic and hydrophilicamino acid residues of linker A with an alternating pattern ofhydrophobic and hydrophilic amino acid residues of linker b, to form astable complex under the conditions effective to promote theinteraction. The interaction may alter the three dimensionalconformation of either or both linkers involved in the interaction andit may also alter the function or activity of either or both linkersinvolved in the interaction. For example, the interaction of linker Awith linker b when linker A and b are part of a monomer as definedherein enhances immunological properties of the resultant monomer.

A method of producing a non-covalently interlinked multimer comprisingthe step of incubating a monomer, (A)-Ag-(a), with a monomer,(B)-Ag-(b), under conditions facilitating peptide interactions to form anon-covalently interlinked multimer is also an aspect of the presentinvention. In this method, Ag, A, a, B, and b are as describedhereinabove.

The Linkers

Linkers may be made up of: a) natural amino acid sequences that bind tonatural amino acid sequences of receptors or vice versa and/or tothemselves, b) custom designed amino acid sequences that bind to othercustom designed amino acid sequences or to themselves because theiramino acid sequences are designed to allow optimal interaction of theirhydrophilic, hydrophobic and neutral amino acid side chains, c) aminoacid sequences that bind in natural proteins to give such proteins theircharacteristic tertiary structure, and/or d) any combinations of these.Amino acid sequences in linkers could be manipulated to increase ordecrease binding affinity and/or enhance presentation of the antigenicepitope. For example, spacer amino acids, such as neutral amino acidsmay be included in the linkers to allow for rotational flexibility andmaximal interaction of a hydrophobic amino acid side chain on one linkerwith a hydrophobic amino acid on another linker and similarly, allowmaximal interaction of a hydrophilic amino acid side chain of one linkerwith a hydrophilic amino acid side chain on another linker. Thus, morestable or less stable multimers could be made with desired degrees offlexibility for binding to receptors.

Linkers are preferably at least 3 amino acids long and not more thanabout 12 amino acids long. The linkers have a pattern of alternatinghydrophobic and hydrophilic amino acids. A preferable pattern is singlyalternating hydrophobic and hydrophilic amino acids, however,alternating sets of 2 or 3 amino acids are also contemplated. Thesealternating sets of hydrophobic and hydrophilic amino acids may beinterrupted by a spacer amino acid as described above to allow maximalinteraction of amino acid side chains. A linker preferably has at leastone hydrophilic and one hydrophobic amino acid.

Modification and changes may be made in the structure of the amino acidlinkers and still obtain a multimer having like or otherwise desirablecharacteristics. For example, certain amino acids may be substituted forother amino acids in a linker without appreciable loss of interactivebinding capacity. Since it is the interactive capacity and nature of anamino acid sequence that defines the linker's functional activity,certain amino acid sequences may be chosen (or, of course, itsunderlying DNA coding sequence) and nevertheless obtain a linker withlike or even countervailing properties (e.g., antagonistic v.agonistic). It is thus contemplated by the inventors that variouschanges may be made in the sequence of a linker (or underlying DNA)without appreciable loss of its ability to multimerize.

Substitution of like amino acids can be made on the basis ofhydrophilicity, particularly where the biological functional equivalentlinker thereby constructed is intended for use in immunologicalembodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference,states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith its immunogenicity and antigenicity, i.e. with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8);tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalentpeptide. In such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginie and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

For purposes of the present invention, hydrophobic amino acids includealanine, valine, isoleucine, leucine, tryptophan and phenylalanine;polar amino acids include histidine, asparagine, glutamine, cysteine,methionine, tyrosine, threonine and serine; positively changed aminoacids include lysine and arginine; negatively charged amino acidsinclude aspartic acid and glutamic acid and a neutral amino acid isglycine. Although not tested, proline was not considered desirable inthe linkers of the present invention since proline promotes a turn inthe secondary structure of a peptide.

Two designations for amino acids are used interchangeably throughoutthis application, as is common practice in the art. Alanine=Ala (A);Arginine=Arg (R); Aspartate=Asp (D); Asparagine=Asn (N); Cysteine=Cys(C); Glutamate=Glu (E); Glutamine=Gln (Q); Glycine=Gly (G);Histidine=His (H); Isoleucine=Ile (I); Leucine=Leu (L); Lysine=Lys (K);Methionine=Met (M); Phenylalanine=Phe (F); Proline=Pro (P); Serine=Ser(S); Threonine=Thr (T); Tryptophan =Trp (W); Tyrosine=Tyr (Y);Valine=Val (V).

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges may be effected by alteration of the encoding DNA; taking intoconsideration also that the genetic code is degenerate and that two ormore codons may code for the same amino acid.

For a particular multimer, amino acids in the linker sequences next tothe desired immunogen, antigen, epitope or ligand may be alteredsomewhat to get optimum activity. In the present examples, the regionclosest to the agent included more neutral amino acids rather thanstrongly hydrophilic or hydrophobic amino acids. These neutral aminoacids are referred to as spacer amino acids.

The Central Component of the Monomer

The central component of the monomer is most readily envisioned as apeptide although various other types of central units may be attached tothe novel peptide linker sequences as described herein. When a preferredcentral component is a peptide, it may be immunogenic, i.e., comprisedesired immunologically active epitopes or combinations of epitopes.These immunoactive epitopes may be, for example: 1) immunosuppressiveepitopes or antigenic epitopes, depending upon the particular end beingsought; or 2) in some cases an activator, stimulator or blocker of adesired biological activity, e.g., a hormone, a hormone receptor orbioactive substance of another type such as activators, stimulators orblockers of any biologic activity in which defined ligands and receptorsare required to interact. When the central component is immunologicallyactive, it should have sufficient size to define the immunologicalactivity, e.g., when it is an antigenic epitope (Ag), it would bind toantibodies. A preferred central component is an HIV HP-6 epitope thatreacts with anti-HP-6 antibody, and having from about 5 to about 30amino acids. In particular, a 13 amino acid HP-6 epitope is mostpreferred, although one of skill in the art would realize in light ofthis disclosure that the epitope may be 6, 7, 8, 9, 10, 11, 12, 14, 15,16, 17, 18, 19, 20, 22, 24, 26 or 28 amino acids long. Thesenon-covalently bound multimers may be used in kits, columns, etc., orenzyme linked immunosorbent (ELISA) assays where the identification,isolation, concentration, or assay of a desired antibody, cell type,ligand, receptor, etc., is required or useful. They may also be used asstabilizers and protectors of labile proteins or other molecules.

The present invention allows the production of potent, synthetic, highmolecular weight, multivalent vaccines made up of large numbers, ortypes of antigenic epitopes. These include glycosylated epitopes orepitopes developed from conserved sequences, from any organism or cell,e.g., viral, bacterial, parasitic, cancer, etc. These same multimerscould also contain ligands that will stimulate signalling molecules,such as hormones, lymphokines and/or cytokines that will enhance theimmune response locally where the multimers are transported and/orprocessed. These same multimers could also contain ligands that wouldbind to FcγR (receptor) on macrophages and dendritic antigen presentingcells, thus greatly enhancing their antigenicity. Targeting of FcγRcells could also be made via monoclonal IgG antibody bound to multimerlinkers.

The synthesis of monomers that include a peptide within their sequenceis readily achieved using conventional peptide synthetic techniques suchas the solid phase method (e.g., through the use of commerciallyavailable peptide synthesizer such as an Applied Biosystems Model 430APeptide Synthesizer). Peptide antigens synthesized in this manner maythen be aliquoted in predetermined amounts and stored in conventionalmanners, such as in aqueous solutions or, even more preferably, in apowder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of immunogenicactivity. However, where extended aqueous storage is contemplated, itwill generally be desirable to include agents including buffers such asTris-HCl or phosphate buffers to maintain a pH of 7.0 to 7.5. Moreover,it may be desirable to include agents which will inhibit microbialgrowth, such as sodium azide or merthiolate. For extended storage in anaqueous state, it will be desirable to store the solutions at 4° C., ormore preferably, frozen. Of course, where the peptide(s) are stored in alyophilized or powdered state, they may be stored virtuallyindefinitely, e.g., in metered aliquots that may be rehydrated with apredetermined amount of water (preferably distilled) or buffer prior touse.

The present invention also provides a method of altering the immunesystem of a subject comprising the steps of i) identifying a subject inneed of immune system alteration; and ii) administering to the subject apharmaceutically effective amount of a non-covalently interlinkedmultimer of the present invention. In a preferred embodiment of thismethod, altering the immune system is stimulating the immune system, thenon-covalently interlinked multimer may be S1 or S2, for example, andcytokine production is induced in the subject. The cytokine may beIFN-τ, IL-1 or INF-α. In a further preferred embodiment of the presentinvention, altering the immune system is suppressing the immune system.This suppression may result from the induction of cytokine IL-b10.Altering the immune system may include bypassing MHC restriction in thesubject.

Vaccines

The present invention contemplates vaccines for use in both active andpassive immunization embodiments. The multimers of the present inventionpossess tertiary structure and have valence and, therefore, areexcellent candidates for providing immune protection for humans oranimals against any organism, cancer, or autoimmune disease. Further,multimers made according to the present invention could be used fordesensitizing hyperimmune individuals, for bypassing majorhistocompatibility restriction, for crosslinking B cell receptors orFcγR receptors, or for better antigen presentation (e.g., linkers may becapable of being cleaved by intracellular enzymes).

Immunogenic multimers, proposed to be suitable for use as a vaccine, maybe prepared in a manner disclosed herein. Preferably, the immunogenicmultimers are extensively dialyzed to remove undesired small molecularweight molecules and/or lyophilized for more ready formulation into adesired vehicle.

The preparation of vaccines that contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables, either as liquid solutions orsuspensions. Solid forms, suitable for solution or suspension in liquidprior to injection, may also be prepared. The preparation may also beemulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines.

To use multimerized peptides that contain immunogenic peptides as thebasis for a human vaccine, the toxicity should be reduced or eliminated.Multimers of the present invention are not mitogenic for naive Tlymphocytes and cannot be considered as superantigens. Superantigens areknown to induce apoptosis or toxicity and therefore would be consideredas harmful components of a vaccine.

Vaccines may be conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10-95% of active ingredient,preferably 25-70%.

The multimers may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the peptide) and those whichare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to theweight of the host.

The multimers of the present invention are particularly advantageous foruse as a vaccine because the valence provided by the multimerizationdecreases the need for an adjuvant. However, further methods ofachieving adjuvant effect for the vaccine include, but are not limitedto, use of agents such as aluminum hydroxide or phosphate (alum),commonly used as 0.05 to 0.1 percent solution in phosphate bufferedsaline, admixture with synthetic polymers of sugars (Carbopol) used as0.25 percent solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between 70° to 101° C. for 30 secondto 2 minute periods respectively. Aggregation by reactivating withpepsin treated (Fab) antibodies to albumin, mixture with bacterial cellssuch as C. parvum or endotoxins or lipopolysaccharide components ofgram-negative bacteria, emulsion in physiologically acceptable oilvehicles such as mannide mono-oleate (Aracel A) or emulsion with 20percent solution of a perfluorocarbon (Fluosol-DA) used as a blocksubstitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the multimer. The assays may be performed by labelingwith conventional labels, such as radionuclides, enzymes, fluorescers,and the like. These techniques are well known and may be found in a widevariety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and3,949,064, as illustrative of these types of assays.

Further Uses of Multimers

Multimers of the present invention are contemplated to be useful in avariety of ways, in addition to their effect on the immune system asexemplified herein. Additional uses that are envisioned include; theactivation or blocking of receptors, the activation of cytokine and/orlymphokine responses, provision of stability for labile molecules, as acarrier, coating or adsorption of toxic drugs or toxins in anyenvironment including treatment of overdoses of toxic drugs, fordetection of antibodies in a biological sample such as ELISA assays, thesynthesis of simple enzymes, protein purification procedures, andantibody capture technology.

A method for detecting anti-HIV antibody in a sample is also an aspectof the present invention. The method comprises the steps of: i)obtaining a sample suspected of having an anti-HIV antibody; ii)contacting the sample with an anti-HIV antibody-binding amount of anon-covalently interlinked multimer of claim 1; and iii) measuringbinding of the multimer to the sample. The presence of anti-HIV antibodyin the sample is determined from the binding. Preferably, the sample ishuman blood. In a preferred embodiment of this method, the multimer isS1 or S2. The measuring step is carried out using a detectably labeledantibody specific for the anti-HIV antibody and the detectably labeledantibody is labelled with a fluorescent tag, a radiolabel or an enzyme.

The present invention also provides a method of producing an antibodyhaving specific binding affinity for the non-covalently linked multimerof the present invention in an animal. This method comprises the stepsof obtaining an animal capable of producing antibody and injecting intothe animal the non-covalently interlinked multimer. Preferably, theantibody is recovered from biological fluid of the animal.

Immunoassay

The present invention, in still another aspect, defines an immunoassayfor the detection of an antibody specific for a multimerized peptide ina biological sample. In one particular embodiment, the immunoassaycomprises; preparing a multimerized peptide, incubating the multimerizedpeptide with the biological sample for a sufficient time to permitbinding between multimerized peptide and antibody present in saidbiological sample, and determining the presence of bound antibody bycontacting the incubate of the multimer and antibody with a detectablylabeled antibody specific for the anti-multimer antibody, wherein thepresence of anti-multimer antibody in the biological sample isdetectable as a measure of the detectably labeled antibody from thebiological sample.

By way of example, the detectably labeled antibody may be labeled withany of a variety of detectable molecular labeling tags, such as; anenzyme-linked (alkaline phosphatase) antibody, a fluorescent-taggedantibody, or a radio-labelled antibody.

A test kit for the detection of HIV antibody in a biological sample isalso an aspect of the present invention. The kit comprises in packagedcombination: i) a carrier means adapted to receive a plurality ofcontainer means in close confinement therewith, ii) a first containermeans including a non-covalently interlinked multimer of the presentinvention, iii) a second container means including a quantity of anunlabelled antibody having specific binding affinity for the multimer,and iv) a third container means including a quantity of a detectablylabelled antibody having specific binding affinity for the unlabelledantibody. Preferably, the detectably labelled antibody is an enzymelinked antibody, a fluorescent tagged antibody, or a radiolabeledantibody. In a preferred embodiment of the test kit, the detectablylabelled antibody is an enzyme linked antibody, and said pack furtherincludes a fourth container means including a quantity of a substratefor the enzyme sufficient to produce a visually detectable product.

Immunodetection Kits

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that themultimers of the present invention may be employed to detect antibodieshaving reactivity therewith. In general, these methods will includefirst obtaining a sample suspected of containing such an antibody,contacting the sample with a multimer in accordance with the presentinvention, as the case may be, under conditions effective to allow theformation of an immunocomplex, and then detecting the presence of theimmunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot, dot blot, indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For diagnostic purposes, it is proposed that virtually any samplesuspected of comprising the antibody sought to be detected, as the casemay be, may be employed. Exemplary samples include clinical samplesobtained from a patient such as blood or serum samples, ear swabs,sputum samples, middle ear fluid, vaginal, or urine samples may beemployed. Furthermore, it is contemplated that such embodiments may haveapplication to non-clinical samples, such as in the titering of antigenor antibody samples, in the selection of hybridomas, in the productionof absorbent columns, kits where any kind of high affinity specificbinding is required, and the like.

In related embodiments, the present invention contemplates thepreparation of kits that may be employed to detect the presence ofantibodies or specific binding agents in a sample. Generally speaking,kits in accordance with the present invention will include a suitablemultimer or antibody directed against such a multimer, together with animmunodetection reagent and a means for containing the antibody orantigen and reagent. The immunodetection reagent will typically comprisea label associated with the antibody or antigen, or associated with asecondary binding ligand. Exemplary ligands might include a secondaryantibody directed against the first antibody or antigen or a biotin oravidin (or streptavidin) ligand having an associated label. Of course,as noted above, a number of exemplary labels are known in the art andall such labels may be employed in connection with the presentinvention.

The container means will generally include a vial into which theantibody, antigen or detection reagent may be placed, and preferablysuitably aliquotted. The kits of the present invention will alsotypically include a means for containing the antibody, antigen, andreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically illustrates the hydropathic score of the amino acidsof monomer M1. Amino acids 1-11 of peptide M1 are the left linker regionwith the sequence: Ser-Arg-Ala-Val-Thr-Ala-Ala-His-Ser-Glu-Ile, SEQ IDNO: 1. Amino acids 12-24 of peptide M1 are the HIV antigen, HP-6, withthe sequence: His-Glu-Asp-Ile-Ile-Ser-Leu-Trp-Asp-Gln-Ser-Leu-Arg, SEQID NO:2. Amino acids 25-35 of peptide M1 are the right linker regionwith sequence: Ser-Glu-Ala-Ile-Ile-His-Val-Leu-His-Ser-Arg, SEQ ID NO:3.

FIG. 2 graphically illustrates the hydropathic score of the amino acidsof monomer M2. Amino acids 1-11 of peptide M2 are the left linker regionwith the sequence: Ala-Leu-Asp-His-Ser-Gly-Arg-Val-Arg-Glu-Thr, SEQ IDNO:4. Amino acids 12-24 of peptide M2 are the HIV antigen, HP-6, withthe sequence: His-Glu-Asp-Ile-Ile-Ser-Leu-Trp-Asp-Gln-Ser-Leu-Arg, SEQID NO:2. Amino acids 25-35 of peptide M2 are the right linker regionwith sequence: Arg-Leu-Ser-Tyr-Asn-Val-Asp-Gln-Met-Arg-Ala, SEQ ID NO:5.

FIG. 3 graphically illustrates the hydropathic score of the amino acidsof monomer S1. Amino acids 1-12 of peptide S1 are the left linker regionwith the sequence: Leu-Lys-Ile-His-Ala-Gln-Ile-Glu-Val-Ser-Val-Ala, SEQID NO:6. Amino acids 13-25 of peptide S1 are the HIV antigen, HP-6, withthe sequence: His-Glu-Asp-Ile-Ile-Ser-Leu-Trp-Asp-Gln-Ser-Leu-Arg, SEQID NO:2. Amino acids 26-37 of peptide S1 are the right linker regionwith sequence: Ala-Asn-Ile-Tyr-Ala-Glu-Ile-Asn-Ser-Tyr-Ile-Arg, SEQ IDNO:7.

FIG. 4 graphically illustrates the hydropathic score of the amino acidsof monomer S2. Amino acids 1-12 of peptide S2 are the left linker regionwith the sequence: Asp-Val-Asn-Ala-Tyr-Leu-Asn-Leu-Arg-Phe-His-Tyr, SEQID NO:8. Amino acids 13-25 of peptide S2 are the HIV antigen, HP-6, withthe sequence: His-Glu-Asp-Ile-Ile-Ser-Leu-Trp-Asp-Gln-Ser-Leu-Arg, SEQID NO:2. Amino acids 26-37 of peptide S2 are the right linker regionwith sequence: Gly-Leu-Gln-Phe-His-Val-Lys-Leu-Tyr-Gly-Glu-Ala, SEQ IDNO:9.

FIG. 5 shows multimeric bands after electrophoresis on an 18%polyacrylamide gel under non-denaturing conditions. Monomers M1, S1, M2,S2 and combinations thereof were tested.

FIGS. 6A-B show gel electrophoretic patterns of multimeric M1 andMultimeric M2. FIG. 6A shows multimeric M1 and multimeric M2 separatedon a 7.5% non-denaturing electrophoretic gel. FIG. 6B shows monomers M1and M2 in the presence of 2% SDS and separated on an 18% polyacrylamidegel.

FIG. 7 shows the proliferative response of lymph node cells from S2immunized ICR mice after exposure to the indicated stimulators.

FIG. 8 shows the proliferative response of lymph node cells from S1immunized BALB/c mice after exposure to the indicated stimulators.

FIG. 9 shows IL-1B induction by treatment of naive human leukocytes withmultimers. The legend is as follows: 1. Control; 2. M1 Stock; 3. M1 Band1; 4. M1 Band 2; 5. M1 Band 3; 6. M1 Band 4; 7. M2 Stock; 8. M2 Band 1;9. M2 Band 2; 10. M2 Band 3; 11. M1 Stock+M2 Stock; 12. M1 Band 1 +M2Band 1; 13. M1 Band 2+M2 Band 2; 14. M1 Band 3+M2 Band 3; 15. M1Left-linker; 16. M1 Right-linker; 17. M1 Left-linker+M1, Right-linker;18. M2 Left-linker; 19. M2 Right-linker; 20. M2 Left-linker+M2,Right-linker; 21. M1 Left-linker+M2, Left-linker; 22. M1Right-linker+M2, Right-linker; 23. M1 Left-linker+M2, Right-linker; 24.M1 Right-Linker+M2, Left-linker; 25. S1 Stock; 26. S2 Stock; 27. S1Stock+S2 Stock; 28. S1 Left-linker+S2, Left-linker; 29. S1Right-linker+S2, Right-linker; 30. S1 Left-linker +S2, Right-linker; 31.S1 Right-linker+S2, Left-linker.

FIG. 10 shows IL-10 induction by treatment of naive human leukocyteswith multimers. The multimer numbers are as in FIG. 9.

FIG. 1 shows TNF-α induction by treatment of naive human leukocytes withmultimers as numbered for FIG. 9.

FIG. 12 shows interferon gamma induction by treatment of naive humanleukocytes with peptide multimers.

FIG. 13 shows binding of antibody in HIV positive serum to S2 peptide asdetermined in an EIA using the indicated quantity of peptide per well.The legend for FIG. 13 is: 3, 20 μg; 2, 10 μg; 1, 5 μg; 4, 1 μg, 5, 0.5μg; 6, HP-6 (20 μg of HP-6); 7, normal serum (20 μg of S2).

FIG. 14 shows binding of antibody in HIV positive serum to S1 peptide asdetermined in an EIA using the indicated quantity of peptide per well.The legend for FIG. 14 is: 3, 20 μg; 2, 10 μg; 1, 5 μg; 4, 1 μg, 5, 0.5μg; 6, HP-6 (20 μg of HP-6); 7, normal serum (20 μg of S2).

FIG. 15 shows binding activity of S1 vs. S2 for HP-6 antibody. Thelegend for FIG. 15 is: , 1 μg/well; □, 5 μg/well.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides custom designed multimeric peptides, themonomers of such multimeric peptides being bound to each other bynon-covalent interactions. By incorporating eight or ten medium tostrongly hydrophobic and hydrophilic amino acids in a binding sequenceor selecting natural amino acid sequences that contained similararrangements of amino acids, the present inventors have linked centralunits such as peptides, using natural binding forces, into large, stablemultimers. Further, the linker sequences were added on both sides of anantigenic epitope greatly enhancing the activity of the epitope.

The present invention teaches the design and synthesis of non-covalentlylinked large peptide multimers that have many of the properties ofbiologically active natural proteins with tertiary structure. Inaddition, these multimers have a considerable advantage over naturalproteins used as vaccines, receptor stimulators or blockers, becausethey have incorporated into them copies of various biologically activehigh affinity epitopes and/or ligands that can activate or block immuneor other important biologic receptors. Multiple copies of epitopes orligands in the same molecule result in multimers with valence. Valenceis necessary for establishing immune responses with memory. It isgenerally known that epitopes in their natural high molecular weightforms, i.e., proteins, are considerably more avid and active than theisolated small molecular weight peptides themselves. Therefore, becausemultimers of the present invention are high molecular weight structures,the activity of the repeated copies of epitopes or ligands incorporatedinto them is enhanced. Biologic studies described herein indicate thatthe affinities, avidities and stabilities are sufficient to inducemorphological changes and cytokine production in naive human leukocytesand an immune response in naive mice. The multimers of the presentinvention may be eluted from single high molecular weight bands onpolyacrylamide gels and reisolated as equally high molecular weightbands on another gel. This is an indication that they are very stable.

No uses of amino acid linker sequences to bind peptides to formbiologically active, non-covalently bound, large molecular weightpeptide molecules or multimers with valence, i.e., repeated copies ofepitopes or ligands, were found in extensive literature and patentsearches of peptide uses for vaccines. U.S. Pat. No. 5,077,195 entitled"POLYPEPTIDES COMPLEMENTARY TO PEPTIDE OR PROTEINS HAVING AN AMINO ACIDSEQUENCE OR NUCLEOTIDE CODING SEQUENCE AT LEAST PARTIALLY KNOWN ANDMETHODS OF DESIGN THEREFOR" concerns the ability of two peptides to bindto each other, one synthesized according to the linear information onthe-strand of DNA that is complementary to the transcribed+strand of DNAor amino acid sequences derived from it, and the other synthesized frominformation on the+strand. The patent enumerated ways that complementarypeptides can be used to induce antibodies useful for the isolation ofreceptors, modulate the immune response by induction of anti-idiotypicantibodies, bind to molecules for their isolation, or identification,and to determine the amino acid sequence of a protein.

No suggestion is made in the literature of using complementary linkerpeptides to make high molecular weight avid multimeric vaccines, nor ofthe fact that multimeric proteins can be made without any informationabout a sequence of codons derived from the genetic or amino acidsequences derived therefrom. Complementary sequences could theoreticallybe used as linkers in the present system, because they can be selectedto contain significant numbers of alternating hydrophobic andhydrophilic amino acid sequences depending on which+strand sequences areselected. However, the experiments described herein indicate that themechanisms given for complementary peptide binding based on the geneticcode actually are not involved in the linkage observed in the presentinvention, especially since 8 of the linkers were picked and/or designedto contain very few complementary amino acids. In addition, when thebinding abilities of peptides that could bind by complementary sequences(M1 and M2, S1 and S2) were investigated by polyacrylamide gelselectrophoresis, the polyacrylamide gel profiles obtained were the sameas for those peptides that did not contain complementary sequences (M1alone, M2 alone, S1 alone, and S2 alone) (see Examples). This findingindicates that, in spite of the design, the binding in all cases wasconsistent with mechanisms of binding associated with a certain amountof alternating hydrophobic and hydrophilic amino acid sequences and notcomplementary binding as defined by U.S. Pat. No. 5,077,195.

The advantages that the present synthetic multimeric vaccines would haveover presently used live virus and killed virus or subunit vaccinesinclude: 1) they would be more stable and not be as risky as live virusvaccines; 2) single or numerous optimally selected, repeating epitopes,could be incorporated into high molecular weight multimers with tertiaryconfirmation, while live virus or killed vaccines only contain one ortwo epitopes of a particular type per molecule; 3) the repeating unitsgive the vaccine molecules valence that would greatly enhance theirability to induce memory cells for long term immunity, thus lessening oreliminating the need for multiple injections; 4) they would be mucheasier and cheaper to produce, therefore, high effective concentrationsof multimeric vaccines could be given, possibly without the use ofadjuvants; 5) multiple epitopes from different virus variants could beincorporated into the same molecule, thus broadening the immune responseto cover infections in which antigenic variants develop; and 6) the samemultimers could contain antigenic determinants as well as ligands thatstimulate lymphokine and/or cytokine responses that enhance immuneresponses.

It was unexpected that peptides containing linkers with only onecomplementary pair of amino acids (in the sense of complementaritydescribed in U.S. Pat. No. 5,077,195) out of 11 would link to eachother, or 3 out of 11 when only two of them are adjacent. It was also asurprise that peptides containing a chimeric linker taken from differentamino acid sequences of ovalbumin/influenza hemagglutinin (OVA/HA) and alinker from myoglobin, both of which bind to MHC product lA^(d) of micewould bind to themselves in multimeric forms (see Examples 1 and 2).

Monomers with linkers complementary to the amino acid sequences OVA/HAand myoglobin would not have been predicted to bind to themselves in asimilar fashion (see Examples 1 and 2). Monomers made from the presentlinkers, in which an antigenic epitope of HIV has been sandwiched, wouldnot have been expected to have biological activity in naive cellcultures of mice and humans and to be immunogenic in mice. Thus, themultimeric peptides of the present invention have in vitro biologicactivity in leukocyte cell cultures and in vivo activity in mice (seeExamples). Another interesting advantage of the present invention isthat epitopes have adjuvant effects from the linkers.

Depending on the size, structural characteristics and ability to inducecytokine by particular molecular weight multimers, they could be eitherimmunogenic (IL-1B, TNFα, IFNγ) or immunosuppressive (IL-10). Studieswith leukocytes are consistent with this, since different forms andnumbers of activated cells appear in cultures based on the multimer orcombination of multimers used to stimulate them (e.g., increased ordecreased incidence of large mononuclear type cells and branchedelongated cells, such as dendritic cells, both of which play activeroles in controlling the immune response). In addition, some multimersdecrease the cell culture effects of others when they are added incombinations, suggesting activation or suppression of leukocyte orimmune responses.

Smaller molecular weight multimers containing epitopes to autoimmuneantigens are expected to be immunosuppressive because of the inverserelationship between size and immunogenicity of many antigens. Thus, theuse of multimers containing these epitopes for potential therapy againstautoimmune disease is feasible.

The multimers may be able to bypass immune histocompatibility complexrestriction, thus broadening the immune response to any restrictedantigen, due to the ability to manipulate and produce a variety of formsof the multimers containing a variety of epitopes.

The present invention also provides for the construction of multimericvaccines containing conformational epitopes derived from the tertiarystructure of antigenic proteins due to the ability to manipulate andproduce a variety of forms of the multimers. Such epitopes are stronglyantigenic.

Due to the ability to manipulate and produce a variety of forms of themultimers containing multiple epitopes (valence), it is also possible tolink B-cell receptors with antigenic epitopes. Thus, it is possible toinduce immune memory cells and also use B-cells as antigen presentingcells. This capability would enhance the potential formation of memorycells, thus decreasing the requirement of yearly vaccination requiredfor effective use of many vaccines.

The present invention provides for multimers containing target aminoacid sequences for enzymes involved in intracellular antigen processingbecause of the ability to manipulate and produce a variety of forms ofthe multimers. These target sequences would be located where the linkersare attached to peptide epitopes. Thus, enhanced cellular processing andpresentation of antigen may be possible.

The multimers of the present invention can be used as stimulators,binders or blockers of any biologic activity or protein molecule whereligands and receptors are required to interact because of the tertiarystructure and potential high affinity of the multimers.

The use of multimers containing specific epitopes for antibody capture,assays, or purification procedures is feasible. Such a method wouldallow isolation, purification, and characterization of antibodies tospecific epitopes that may be highly important for the protection ofanimals and humans against virus, bacterial, parasitic, cancer, and thelike; thus enabling refined design of epitopes to be included invaccines.

The present invention also provides for the production of syntheticvaccines that are less likely to induce autoimmune disease due to theability to make linkers that could mimic normal beta sheet amino acidsequences of proteins found in an animal species, or sequences that arenot found in a species' proteins.

Due to the way the linkers bind, it would be possible to producemultimers with biological activity by placing the epitope(s) orligand(s) at the end of a linker or sequence of linkers. Thus, epitopesor ligands could be made to extend from the multimers. These could bemore effective in binding to certain receptors for activating orblocking purposes.

Due to their tertiary structure and potential high avidity and affinity,multimers containing a desired ligand could be used as improved bindersof any protein molecule, or part of a protein molecule, in which aligand and its receptor can interact, e.g., in kits, columns, ELISAassays or the like, where the identification, isolation, concentration,or assay of a desired antibody, cell type, ligand, receptor, or the likeis required or useful. Multimers of the present invention may stabilize,protect, and/or carry labile proteins or adsorb toxic molecules.

The present invention also envisions the production of synthetic enzymesout of multimers because of the ability to manipulate and produce avariety of forms of the multimers. The multimers of the presentinvention may also be used in injectable, oral, anal, vaginal and/ornasal vaccines, and the like.

Further applications for the immunoenhancing linker systems of thepresent invention are: (1) enhanced immunogenicity of conserved epitopesof HIV or other organisms, (2) induction of immunogenicity for desiredamino acid sequences on the surface of HIV or other organisms, (3)enhanced immunogenicity of tumor antigens, (4) desensitization forallergens, (5) cell migration, and (6) wound healing (induction ofcytokines, growth factor, etc. for enhanced healing).

Further applications for immunosuppressive linker systems would be: 1)improvement in treatment of autoimmune disease, and 2) prevention ofapoptosis in treatment of early AIDS.

Multimers may be attached to multimers containing linked foreignepitopes, e.g., tumor antigen, virus antigens such as mumps virus, andthe like, to virus infected cells or cancer cells, thus making them muchmore immunogenic and/or susceptible to killing by specific antibodies orcytotoxic cells directed toward the epitope. Attachment could beaccomplished by: a) linking multimers to monoclonal antibodies thatreact with specific ligands on the cells; b) linking multimers toprotein receptors, or ligands that will bind to a specific uniquereceptor on a cell; c) incorporating specific parts of ligands orreceptors into multimers that bind to their counterparts.

Since the monomers that make up the multimers can bind under naturalconditions, it may be possible to produce and use multimers inrecombinant technology including: a) the production of multimericvaccines; or b) the use of recombinant vectors producing multimers asvaccines. The DNA sequence of any monomer can be easily determined fromthe genetic code and these sequences are sufficiently small to putseveral into the same vector, thus it may be possible to expressmultimers in vectors such as plasmids or phage using bacteria or virusesas hosts, for example.

Since multimers bind in a variety of ways, the production of multimersthat can bind to labile proteins, vaccines, hormones, and the like, andmake them more stable, protect them, or serve as carrier proteins isenvisioned by the present invention. Further, multimers that canspecifically block undesired nonspecific binding, e.g., binding toprotein isolation and purification equipment, containers, contaminatingproteins, and the like, are envisioned by the present invention.Multimers that can absorb or neutralize toxic materials in blood, e.g.,drugs or by-products from pharmaceutical agents are also envisioned.

Due to the ability to manipulate and produce a variety of forms of themultimers containing a variety of ligands, it is envisioned toincorporate ligands into multimers that would facilitate their entryinto cells, e.g., virus components. Such ligands incorporated along withenhancer or blocker ligands could help control intracellular processesincluding processes that lead to tumorigenicity, apoptosis, and thelike. The ability of the multimers themselves to cause autoimmunedisease should be very low because the amino acid sequences used can bechosen to exclude any known vertebrate sequence.

The following examples are meant to illustrate particular preferredembodiments of the present invention, in addition to a description ofthe general approach involved. These examples are not meant to limit thescope of the claimed invention but merely to illustrate the preferredembodiments.

EXAMPLE 1 DESIGN OF LINKER-Antigen-LINKER MONOMERS

Monomers were designed and prepared that had amino acid linker sequencesare bound to the N- and C-terminal ends of a peptide antigenic epitope.Thus, a monomer is designated linker-antigen-linker. A linker sequenceis an amino acid sequence that would non-covalently bind to an aminoacid sequence on the end of another monomer or possibly to itself due toa pattern of alternating hydrophobic and hydrophilic amino acidresidues. In the present example, the antigen is a peptide and iscovalently bound to the linkers, i.e., the antigen is sandwiched in themiddle of a set of linkers.

In the following illustration, the linker AAAAAAA was designed to linkwith linker bbbbbbbb and not with linker aaaaaaaa; and the linkeraaaaaaaa was designed to link with BBBBBBBB and not AAAAAAAA. In thismodel, the letters represent specific amino acid sequences that weredesigned to link, and Ag stands for an antigenic antigen or ligand.##STR1##

Unexpectedly, results also indicated that linkers of a monomer alsobound to each other as shown in this illustration: ##STR2##

Specific amino acid linkers made by the present inventors andincorporated into monomers are listed in Table 1. Those linkerspositioned to the left or at the amino terminal end of the antigen aredesignated L, e.g., M1-L; and those linkers positioned to the right orat the carboxy terminal end of the antigen are designated R, e.g., M1-R.The amino acids are designated as hydrophobic, Φ; polar, δ; charged,+/-; or neutral, 0.

For purposes of the present invention, hydrophobic amino acids includealanine, valine, isoleucine, leucine, tryptophan and phenylalanine;polar amino acids include histidine, asparagine, glutamine, cysteine,methionine, tyrosine, threonine and serine; positively changed aminoacids include lysine and arginine; negatively charged amino acidsinclude aspartic acid and glutamic acid and a neutral amino acid isglycine. Proline is not desirable in the linkers of the presentinvention since proline promotes a turn in the secondary structure of apeptide.

                                      TABLE 1                                     __________________________________________________________________________    Amino Acid Sequence of Linkers Showing Patterns of Hydropathy.sup.1           __________________________________________________________________________    M1-L                                                                              Ser                                                                              Arg                                                                              Ala                                                                              Val                                                                              Thr                                                                              Ala                                                                              Ala                                                                              His                                                                              Ser                                                                              Glu                                                                              Ile   SEQ ID NO: 1                              δ                                                                          +  Φ                                                                            Φ                                                                            δ                                                                          Φ                                                                            Φ                                                                            δ                                                                          δ                                                                          -  Φ                                       M1-R                                                                              Ser                                                                              Glu                                                                              Ala                                                                              Ile                                                                              Ile                                                                              His                                                                              Val                                                                              Leu                                                                              His                                                                              Ser                                                                              Arg   SEQ ID NO: 3                              δ                                                                          -  Φ                                                                            Φ                                                                            Φ                                                                            δ                                                                          Φ                                                                            Φ                                                                            δ                                                                          δ                                                                          +                                           M2-L                                                                              Ala                                                                              Leu                                                                              Asp                                                                              His                                                                              Ser                                                                              Gly                                                                              Arg                                                                              Val                                                                              Arg                                                                              Glu                                                                              Thr   SEQ ID NO: 4                              Φ                                                                            Φ                                                                            -  δ                                                                          δ                                                                          0  +  Φ                                                                            +  -  δ                                     M2-R                                                                              Arg                                                                              Leu                                                                              Ser                                                                              Tyr                                                                              Asn                                                                              Val                                                                              Asp                                                                              Gln                                                                              Met                                                                              Arg                                                                              Ala   SEQ ID NO: 5                              +  Φ                                                                            δ                                                                          δ                                                                          δ                                                                          Φ                                                                            -  δ                                                                          δ                                                                          +  Φ                                       S1-L                                                                              Leu                                                                              Lys                                                                              Ile                                                                              His                                                                              Ala                                                                              Gln                                                                              Ile                                                                              Glu                                                                              Val                                                                              Ser                                                                              Val                                                                              Ala                                                                              SEQ ID NO: 6                              Φ                                                                            +  Φ                                                                            δ                                                                          Φ                                                                            δ                                                                          Φ                                                                            -  Φ                                                                            δ                                                                          Φ                                                                            Φ                                    S1-R                                                                              Ala                                                                              Asn                                                                              Ile                                                                              Tyr                                                                              Ala                                                                              Glu                                                                              Ile                                                                              Asn                                                                              Ser                                                                              Tyr                                                                              Ile                                                                              Arg                                                                              SEQ ID NO: 7                              Φ                                                                            δ                                                                          Φ                                                                            δ                                                                          Φ                                                                            -  Φ                                                                            δ                                                                          δ                                                                          δ                                                                          Φ                                                                            +                                        S2-L                                                                              Asp                                                                              Val                                                                              Asn                                                                              Ala                                                                              Tyr                                                                              Leu                                                                              Asn                                                                              Leu                                                                              Arg                                                                              Phe                                                                              His                                                                              Tyr                                                                              SEQ ID NO: 8                              -  Φ                                                                            δ                                                                          Φ                                                                            δ                                                                          Φ                                                                            δ                                                                          Φ                                                                            +  Φ                                                                            δ                                                                          δ                                  S2-R                                                                              Gly                                                                              Leu                                                                              Gln                                                                              Phe                                                                              His                                                                              Val                                                                              Lys                                                                              Leu                                                                              Tyr                                                                              Gly                                                                              Glu                                                                              Ala                                                                              SEQ ID NO: 9                              0  Φ                                                                            δ                                                                          Φ                                                                            δ                                                                          Φ                                                                            +  Φ                                                                            δ                                                                          0  -  Φ                                    S20-L                                                                             Arg                                                                              Phe                                                                              His                                                                              Tyr                        SEQ ID NO: 10                             +  Φ                                                                            δ                                                                          δ                                                          S20-R                                                                             Gly                                                                              Leu                                                                              Gln                                                                     0  Φ                                                                            δ                                                             S22-L                                                                             Leu                                                                              Arg                                                                              Phe                                                                              His                                                                              Tyr                     SEQ ID NO: 11                             Φ                                                                            +  Φ                                                                            δ                                                                          δ                                                       S22-R                                                                             Gly                                                                              Leu                                                                              Gln                                                                              Phe                        SEQ ID NO: 12                             0  Φ                                                                            δ                                                                          Φ                                                            __________________________________________________________________________     .sup.1 L -- left linker, R -- right linker, Φ -- hydrophobic amino        acid, δ -- polar amino acid, +/- -- positively/negatively charged       amino acid, 0 -- neutral amino acid.                                     

The linkers for monomer M1 (Table 1) were chosen from amino acidsequences determined by Sette et al. to bind strongly to mouse MHCIA^(d) molecules. Thus, it was known that these sequences have anendogenous binding capacity and eight or nine hydrophobic andhydrophilic residues each. The 11 amino acid sequence chosen for theleft linker (M1-L) contained essentially the same sequence as Sette'schimeric peptide OVA/HA 5.9 (OVA 324-334/HA 132-142), except that anarginine residue was substituted for glutamine at position 2. The aminoacid sequence chosen for the right linker contained a myoglobinsequence. Since the linkers were made up of amino acid sequences fromdifferent proteins and since they both bound to MHC IA^(d) molecules, itwas thought highly unlikely that they would bind to each other. Thus,the M1 linkers were expected to fit the non-interacting selectivecriteria for left and right linkers on the same monomer.

The linkers on M2 (see Table 1) were designed to be complementary to thelinkers on M1, using the Blalock et al. theory (Blalock et al., Bost etal.), (i.e., amino acid sequences consistent with the probable geneticcode of the non-transcribed DNA strand) with the exception that, in theleft linker, Asp was placed opposite of Ala in position 3 and Thr wasplaced opposite of Ile in position 11. These substitutions were made sothat the linker would be somewhat more hydrophilic on the N-terminus andsomewhat more neutral on the end attached to the antigen. Again thedesign of these monomers made it unlikely that the linker sequences onthe N-and C-terminal ends of the monomer M2 would bind to each other.

Hydrophobic and hydrophilic binding between beta (β) sheet structures,and/or alpha (α) helix structures are very important for folding andstabilizing natural proteins. However, in most natural proteinstructures, usually only 2 to 4 alternating hydrophilic and hydrophobicresidues are present in α helices or β sheets that can interact.Therefore, another pair of monomers, S1 and S2, were specificallydesigned to allow optimal interaction between hydrophobic andhydrophilic residues, i.e., in contrast to the M1 linkers, the left andright linker amino acid sequences of S1 and S2 were not based orselected from any known sequence of amino acids. In S1, an extendedsequence pattern of alternating hydrophobic and hydrophilic amino acidswas selected. The N- and C-terminal linker sequences of S1 and S2 (seeTable 1) also were designed not to bind to each other according to theBlalock theory. Thus, of the 12 amino acids in each linker, no more thanthree were complementary, indicating that it would be highly unlikelythat the monomer would bind to itself in a manner described by Blalocket al.

Specifically, monomers S1 and S2 were designed to contain linkersequences having alternating strong to intermediate hydrophobic andcharged or polar hydrophilic amino acid residues (Table 1). Startingwith the N-terminal amino acid of the left and right linkers, aminoacids with strong to intermediate hydrophobic side chains were placedopposite amino acids with a comparable hydrophilic side chain to give analternating pattern in the S1 versus S2 linkers. Thus, stronglyhydrophobic amino acids in the linkers of the S1 peptide could bind withstrongly hydrophobic amino acids in the S2 linkers and vise versa. Thehydrophilic amino acids were similarly situated so they could bind witheach other. More neutral or polar amino acids were placed at positions10 and 34 of S1 and a neutral Gly at 26 and 35 of S2 to enableflexibility. One of skill in this art would realize in light of thepresent disclosure that the amino acids in the linker sequences next tothe desired epitope or ligand may have to be altered to get optimumactivity. It was expected that peptides having these amino acid sequencepatterns would bind to each other or themselves in a fashion similar toparallel beta sheets. Thus, the alternating nature of the hydrophobicand hydrophilic amino acids in the left and right linkers of the samepeptide would allow binding of the same peptides to each other to formmultimers containing one type of monomer.

Two further monomers, S20 and S22, were synthesized to examine theminimum number of alternating hydrophobic and hydrophilic (or polar)amino acids needed in linkers to confer biological activity to a monomerof which the linkers are a part. Monomer S20 contained the HP-6 epitope(described below) sandwiched between linkers that contained onehydrophobic amino acid, and monomer S22 contained HP-6 sandwichedbetween linkers that contained two hydrophobic amino acids. Thus, S20has one and S22 has two alternating hydrophobic amino acids at each endof the epitope.

The human immunodeficiency virus (HIV) T cell epitope, peptide HP-6, wasselected from Hale et al. (1989) and incorporated into each monomerbetween the left and right linkers. This particular gp160 envelopeepitope (residues 112-124) was chosen because it was immunogenic inH-2^(k) haplotype mice but not in H-₂ ^(d) haplotype mice. Thus, itcould be used to study the immunogenicity of multimers containing HP-6in restricted H-2^(d) mice. The sequence of HP-6 is:

    His-Glu-Asp-Ile-Ile-Ser-Leu-Trp-Asp-Gln-Ser-Leu-Arg, SEQ.ID.NO.2

The hydropathic patterns of the monomers M1, M2, S1, and S2 are shown inFIG. 1-FIG. 4.

The above-described peptides were synthesized by the solid phase methodof Merrifield (1963) using an automatic peptide synthesizer, withstandard t-butoxycarbonyl (t-Boc) chemistry that is well known to thoseskilled in this art in light of this disclosure. The amino acidcomposition and the sequence analysis of the synthesized peptidescorrespond to the expected compositions. The amino acid composition ofthe peptides was determined by amino acid analysis. The purity of thepeptides was determined by sequence or HPLC.

EXAMPLE 2 GEL PROFILES OF MULTIMERS

To determine if the monomers would bind to themselves or to each otherand form multimers, they were solubilized in 100 percent DMSO anddiluted to 1.0 to 2.0 mg/ml with TRIS-HCl glycine buffer at pH 8.8.Aliquots (50 μl) of individual monomers and combinations of monomerswere then subjected to non-denaturing polyacrylamide gel electrophoresis(PAGE) on 18 or 7.5 percent gels, pH 8.8, or to denaturing sodiumdodecyl sulfate (SDS) PAGE. Following electrophoresis, the peptides inthe bands of the gels were visualized with Coomassie blue and/or silverstains.

The results of two representative experiments on 18 percentnon-denaturing gels (FIG. 5) showed that certain monomers bound to eachother to form multimers that migrated as distinct bands. The sharpnessand equity of distance between the bands indicated that the monomers hadbound or complexed to form multimers in a very distinct, repetitive, andstable manner. If binding had been random, the multimers would haveappeared as long gray smears. Each band represents a multimer with adifferent molecular weight. The precise molecular weights of themultimers are not defined because of the difficulty in determining themolecular weights of proteins electrophoresed in non-denaturing gels.However, it is apparent from the positions on the gels that themultimers have considerably higher molecular weights than the monomerssince the monomers migrated close to the dye front when treated with 2%SDS and electrophoresed in a 7.5% non-denaturing gel (FIG. 6A) orsubjected to denaturing SDS PAGE in an 18% gel (FIG. 6B). The broadnessof the bands at the bottom of the denaturing gel indicate that, evenunder these conditions, some binding occurred that was stable.Importantly, these findings indicate that the multimers are made up ofsequentially repeating monomers resulting in multimers with a gradientof different sizes. An estimation of a molecular weight for the smallermultimers is greater than about 15,000 since multimers M1, M2, S1, S2,M1+M2, and S1 +S2 did not diffuse out of dialysis tubing having a M_(r)cutoff size of 12,000 to 15,000.

More specifically, it can be observed in FIG. 5, FIG. 6A and FIG. 6Bthat the M1 monomer complexed to form four bands of multimers while theM2 monomer formed three. Some intermediate random binding also occurredwith M1, as indicated by the diffuse gray areas. It is possible that theM1 monomer is more likely to form higher molecular weight multimers.Although the two monomers differ considerably in their amino acidsequences (the linker sequences of the M2 peptide is the complement ofM1 according to the Blalock et al. theory) the multimers of bothessentially migrate the same. Thus, similar mechanisms of binding andformation of multimers are probably operating in both instances. When M1and M2 were mixed and separated by electrophoresis, the pattern ofmigration of the multimers were the same as M1 by itself. Thus, it ispresently not possible to determine whether M1 and M2 bound to eachother. However, if they did bind, the banding pattern suggests that themechanism of binding was similar to the binding of M1 and M2 tothemselves. Thus, the binding is not consistent with a complementarytype of "lock and key binding" as proposed by Blalock et al., since itis highly unlikely that monomers M1 and M2 would bind to themselves by"lock and key" type binding.

When M1 and M2 were mixed with S1 or S2 and separated byelectrophoresis, the migration patterns were again the same as M1 or M2alone. Thus, it is not possible to tell whether binding occurred betweenthese monomers. Only slight binding appeared to occur with the S1, S2and S1 plus S2 monomers, however, where bands can be observed, thepatterns are the same as with the M1 and M2 monomers, again suggesting asimilar mechanism of binding. When higher concentrations of S1 and S2were mixed and separated by electrophoresis, the bands were much moredistinct. Additionally, the S1 and S2 multimers do not stain well withthe silver stain.

Taken together, the data indicate that whether individual multimers orany combination of multimers were electrophoresed on non-denaturingpolyacrylamide gels, the molecular sizes observed were essentially thesame. This data strongly indicate that the fundamental mechanism offormation of all the multimers is the same.

The actual length or minimum number of strongly hydrophobic orhydrophilic amino acids required to get stable binding and/or biologicalactivity per linker is estimated as follows. Both linking and biologicalactivity were observed for the M1 and M2 multimers as described in laterExamples. Since these multimers have as few as two to three stronglyhydrophobic or hydrophilic amino acid residues per linker that couldinteract with each other, these numbers approximate the minimum numberof alternating strongly active amino acids required for binding andenhanced activity. The HIV epitope without linkers has no in vitroactivity and very little, if any, in vivo activity.

To confirm the stability of the multimers in the bands and to assurethat the multimers elutable from the different bands would remainstable, multimers of M1 and M2 were eluted from the precise areas of thegel and re-subjected to non-denaturing PAGE. The bands were resolvedwith Coomassie blue followed by silver stain. The results show that themultimers migrated to form single bands at the same relative positionsas they were eluted from the first gel. None of the bands dissociatedinto lower band forms; indicating that the multimers are stable.Together, these data indicate that the eluted multimers are both stableand homogeneous and that the multimeric forms used to immunize mice orstimulate leukocyte cultures in the following Examples were of the samesize as the ones eluted from the gels.

In summary, S1, S2, M1 and M2 monomers all can form distinct bands ofsimilar types of multimers on gels. It appears that the monomers mustbind together in several (3-4, depending on the linkers)thermodynamically stable conformations. Although the S monomers do notform distinct multimers on low percent gels, the distribution, i.e.,size, of the multimers on the higher percentage gels are similar to theM multimers but more random. The differences between the two M and Smultimers are that the S multimer stocks are much more active in thesystems tested in the following Examples than the M multimer stocks.This is an advantage because these multimers could be used withoutextensive isolation procedures and, of course, could be incorporatedinto vectors for commercial, e.g., vaccine and other biological uses.The ability to form multimers of different sizes and possibleconformations allows for diversity for optimizing presentation ofligands or epitopes.

EXAMPLE 3 IMMUNIZATION OF MICE

The present example provides data demonstrating that specific andgeneral immune responses were elicited in mice due to injection ofmultimers of the present invention.

M2 multimers (containing HP-6) were eluted from the upper two bands ofnon-denaturing PAGE gels and studied to determine if they would differfrom: 1) each other in their ability to stimulate immune responses invivo, or 2) stock M2, which contains all the multimers, or 3) the HIVepitope alone. Specifically, female ICR outbred mice were inoculatedsubcutaneously at the base of the tail with the following samples: 50 μgof upper band (band 1 of FIG. 6A), lower band (band 2 of FIG. 6A),multimers, M2 dialyzed stock, or the HIV epitope. Each inoculum wasthoroughly emulsified in Freund's complete adjuvant prior to injection.The amount of stimulation, indicated by ³ H-thymidine incorporation intolymph node cells (4×10⁶ /ml) prepared seven days after immunization andincubated for five days in the presence of the indicated stimulants, isshown in Table 2. Staphylococcal enterotoxin A (SEA), a specific T cellmitogen, was also used to stimulate cultured cells to determine if Tcell activity had been generally stimulated.

                                      TABLE 2                                     __________________________________________________________________________    T Lymphocyte Stimulation Following Immunization of ICR                        Mice with Multimers.sup.a                                                     Lymph Node                                                                             Mice Immunized With:                                                 Cells Stimulated                                                                       M-2 Stock                                                                            HIV-epitope                                                   with:    Dialyzed                                                                             alone  M2-Band 1                                                                            M2-Band 2                                       __________________________________________________________________________    Media    1397 ± 283                                                                        1189 ± 433                                                                         771 ± 167                                                                         1844 ± 387                                  SEA      11243 ± 2405                                                                      5318 ± 732                                                                        9096 ± 2193                                                                       249396 ± 20925.sup.b                         0.2 μg/ml                                                                  M-2 Stock                                                                     Dialyzed                                                                      8 μg/ml                                                                             1611 ± 524                                                                         3498 ± 1743                                                                      5216 ± 2476.sup.c                                                                  29682 ± 4277.sup.b                          4 μg/ml                                                                             1511 ± 270                                                                         1456 ± 1328                                                                      2152 ± 209.sup.b                                                                   24062 ± 6149                                2 μg/ml                                                                             3613 ± 228                                                                         861 ± 535                                                                        1912 ± 425                                                                         19135 ± 184                                 1 μg/ml                                                                             N.D.   N.D.   N.D.    14168 ± 5828                                M-2 Band 1                                                                    8 μg/ml                                                                             1059 ± 10                                                                         1113 ± 70                                                                         1097 ± 359.sup.c                                                                   6264 ± 1276.sup.b                           4 μg/ml                                                                              2664 ± 1143                                                                       697 ± 236                                                                         901 ± 28                                                                          8725 ± 1537                                 2 μg/ml                                                                             2263 ± 713                                                                        499 ± 28                                                                           756 ± 151                                                                         7244 ± 2815                                 1 μg/ml                                                                             N.D.   N.D.   N.D.    6979 ± 1276                                 M-2 Band 2                                                                    8 μg/ml                                                                             2457 ± 261                                                                         926 ± 262                                                                        1384 ± 96.sup.c                                                                    13089 ± 424.sup.b                           4 μg/ml                                                                              1992 ± 1150                                                                       640 ± 148                                                                        1011 ± 41                                                                          9746 ± 4460                                 2 μg/ml                                                                             1535 ± 38                                                                         827 ± 51                                                                           749 ± 160                                                                         9391 ± 1932                                 1 μg/ml                                                                             N.D.   N.D.   N.D.    6437 ± 1640                                 HIV-epitope alone                                                             8 μg/ml                                                                             786 ± 30                                                                           1493 ± 1109                                                                      5047 ± 3941                                                                        5863 ± 386.sup.b                            4 μg/ml                                                                             1870 ± 199                                                                         714 ± 255                                                                        2114 ± 420.sup.c                                                                   6523 ± 670                                  2 μg/ml                                                                             1318 ± 690                                                                        1003 ± 680                                                                        1332 ± 95                                                                          4291 ± 389                                  1 μg/ml                                                                             N.D.   N.D.   N.D.    4205 ± 540                                  __________________________________________________________________________     .sup.a = cpm ± S.D., .sup.3 Hthymidine was added to cultures on day 4      and amounts incorporated in DNA counted on day 5.                             .sup.b = Significant at the p = <0.05 level by the Student's ttest.           .sup.c = Significant at the p = 0.1 level                                

The results indicate that immunization with the lower band (Band 2)multimer greatly enhanced the lymph node cell response to SEA as well asthe response to the dialyzed M-2 stock, the upper band (Band 1)multimers, and to itself (P-<0.0001, 0.045 and 0.016, respectively, byStudent's t test). Importantly, the response to the HIV-epitope alonewas significant at the P=0.043 level.

In contrast, lymph node cells of mice immunized with the Band 1multimers did not respond as significantly to Band 1 or Band 2 multimers(P=0.095). However, a significant response to SEA occurred (P=<0.05). Nosignificant responses occurred when mice were immunized with the HIVepitope alone or dialyzed stock, which theoretically contains all themultimers. This observation is consistent with the findings of othersthat find animals immunized with peptides alone, or even peptides boundto carrier protein, often do not respond very strongly.

It is noteworthy that a significant response occurred to the HIV epitopealone when the Band 2 multimer was used as antigen (P=0.043), indicatingthat specific immunoactivation of lymph node cells, very likely T cells,to the HIV epitope occurred. The lymph node response to the HIV epitopein mice immunized with the Band 1 was marginally significant at theP=0.1 level; however, a good dose response occurred that, whenstatistically evaluated, should determine this response as significant.It presently has not been determined whether a specific immune responseto the linker part of the M2 multimer occurred. It is possible that someor all of the positive stimulation responses to the dialyzed stock, Band2 and Band 1 were specific for the HIV epitope and the degrees ofdifferences observed were due to the various ways epitopes in themultimers were presented to the immune system and/or to the sensitizedcells stimulated during culture. Thus, dialyzed stock containingnumerous forms of the multimers was an excellent stimulator of lymphnode cell cultures prepared from mice immunized with the Band 2multimers, but a poor stimulator of the immune system when it was usedas antigen.

Overall these studies indicate that multimers designed according to thepresent invention can be effectively used to stimulate specific and"general" immune responses, as indicated by the enhanced immuneresponses to the HIV epitopes present in multimers and to SEA.

The immunologic effects of the S1 and S2 multimers in mice have alsobeen tested. Because of the inability to stain bands intensely on gelswith these monomers, which also "sandwich" an HIV epitope, stockpreparations were used to immunize groups of ICR, BALB/c and C3H mice.Immunizations, lymph node cell preparations, and stimulation assays wereall performed as described in this Example.

The proliferative responses and standard deviations of cultured lymphnode cells from ICR outbred mice immunized with S2 stock afterstimulation with the indicated multimers and monomers are shown in FIG.7. The S1 peptide was not immunogenic in these mice. The stimulationindex (y axis) was obtained by dividing the radioactive counts ofthymidine incorporated into stimulated cultures prepared from immunizedmice by the counts incorporated into stimulated cultures fromunimmunized mice. It can be observed that the S2 stock multimers, whichcontains all forms of the multimers, was highly immunogenic in ICR micewhen compared to the other multimers and its own linkers (S2LL+RL).Stimulation by S1, which contains the HIV epitope but different linkerswas twice as active as the other peptides used, indicating that the miceimmunized with S2 had responded, at least in part, to the HIV epitope.This specificity is further indicated by the ability of HP-6, the HIVepitope alone, to stimulate the cultures from mice immunized with S2compared to the HP-6 activity in cultured cells taken from miceimmunized with HP-6 (HP-6 vs. HP-6). Unexpectedly, the linkers alone hadactivity even when mice had not been exposed to them, e.g., the S1LL+RL. Stimulation by the M1 and M2 stock multimers was less thanexpected but at least equivalent to PPD which should be active becauseFreund's complete adjuvant was used in the immunizations. Overall thedata suggest that the S2 stock multimers are highly immunogenic in ICRmice and that the responses are, at least in part, specific for the HIVepitope HP-6. The fact that some linkers are active in cultured lymphnode cells from mice not previously exposed to them suggests that theyhave immunomodulatory effects themselves. Since the S1 stock multimerswere not immunogenic in these mice, these multimers may be restricted inthis strain of mouse or may be immunosuppressive.

FIG. 8 shows the proliferative responses and standard deviations oflymph node cells from BALB/c inbred mice. This strain of mouse wasinvestigated because it had been reported that HP-6 was immunologicallyrestricted in mice that had been immunized with gp160 envelope proteinof HIV (Hale, et al.). Mice were immunized with S1 or S2 multimers andthe lymph node cells stimulated as indicated. These mice could beimmunized with the S1 stock but not the S2, possibly because S2 wasimmunosuppressive in these mice (Table 3).

                                      TABLE 3                                     __________________________________________________________________________    Immunosuppression of the Proliferative Response                               of Lymph Node Cells from Mice Immunized with the                              S2 Peptide and Stimulated by Media, PPD or SEA                                Mice immunized with:                                                          Mouse                                                                             S1 peptide + FCA.sup.a                                                                   S2 peptide + FCA                                                                          FCA alone                                          strain                                                                            Media                                                                             PPD.sup.b                                                                        SEA.sup.c                                                                         Media                                                                             PPD SEA Media                                                                             PPD                                                                              SEA                                         __________________________________________________________________________    ICR  8.sup.d                                                                          6639                                                                             4660                                                                              34  11041                                                                             14333                                                                             16  1535                                                                             115                                         BALB/c                                                                            30  717                                                                              4346                                                                              6     24*                                                                               53*                                                                             10  209                                                                              1528                                        C.sub.3 H                                                                         21  1903                                                                             12333                                                                             9    253                                                                               8157                                                                             4   33 2567                                        __________________________________________________________________________     .sup.a = Freunds complete adjuvant                                            .sup.b = Purified protein derivative of M. tuberculosis                       .sup.c = Staphylococcal enterotoxin A (T cell mitogen)                        .sup.d = Counts per minute                                                    * = Significant suppression (p = <.001 by student test)                  

Thus the response in BALB/c mice was almost opposite of that observedfor the ICR mice and shown in FIG. 7. The differences may be due torestriction and/or immunosuppression mediated by the S2 peptides in thisstrain of mice. Some specificity of the response to the HIV epitope isindicated from the difference of the response to itself and its linkers(17.9 vs 4.9). All the other responses were substantial and similar tothe response mediated by PPD. These data indicate that the HIV epitopepresented in the form of S1 multimers was not restricted and that the S1peptide multimers broadly stimulated immune activities. This approachmay bypass MHC restriction.

Table 4 shows the abilities of S20 and S22 to boost the proliferativeresponse in lymph node cultures prepared from S2 immunized ICR nice. Theincreases were significant (P<0.001) and size related: S22>S20>Hp-6.Since all of the linker sequences are missing from HP-6 and most of themfrom S20 and S22, the data strongly suggest that the lymph nodeproliferative responses are specific for HP-6. Further, if the size ofHP-6 is increased by adding two or four alternating hydrophobic andhydrophilic amino acid sequences, their ability to stimulate the lymphnode cells is increased. In the same experiment, the S20 and S22peptides inoculated into mice were not able to induce a proliferativeresponse in mice. Thus, the linkers associated with HP-6 must be largerthan two alternating hydrophobic or hydrophilic amino acid sequences inorder to function as immunogens.

                  TABLE 4                                                         ______________________________________                                        Proliferation of Lymph Node Cell Cultures                                     Prepared from ICR Mice Immunized with S1 or S2 Peptides                       and Stimulated with Truncated Forms of S2.                                    Lymph Node                                                                    Cultures (CPM × 6)                                                      Stimulated                                                                             ICR Mice Immunized with:                                             with:    Tris-HCl FCA       S1 + FCA                                                                             S2 + FCA                                   ______________________________________                                        Media    59.89    215.11    79.22  517.11*                                    S1 Stock 73.67    114.33    126.33 240.56                                     S2 Stock 46.78    174.11    86.33  1884.22*                                   HP-6     97.11    179.78    84.22  1053.44*                                   S20      179.11   421.89    61.22  2492.22*                                   S22      172.11   466.11    97.56  3464.00*                                   PPD      90.11    254.67    54.89  380.89                                     SEA      3383.78  3288.22   4984.56                                                                              3772.11                                    ______________________________________                                         * = Significantly different from controls and each other (P < 0.001 by        student's t test).                                                       

Table 4 also shows that the S1 peptide not only is non-immunogenic inICR mice, but actually suppressed all the proliferative responsesinduced by culture stimulators with the exception of the S1 stock andSEA. Thus, depending on the allotype of the mouse (S1 is immunoenhancingin BALB/c mice, FIG. 19) multimeric peptides may be immunoenhancing orimmunosuppressive. Application of immunosuppressive activities could bevery important for suppression of autoimmune diseases, transplants,allergies, etc.

EXAMPLE 4 THE EFFECTS OF MULTIMERS ON LEUKOCYTE MORPHOLOGY

The present Example describes the different effects of multimers onleukocyte morphology.

Cultures prepared from Ficoll-hypaque enriched human leukocytes (3×10⁶/ml) were treated with the indicated M1, M2, S1, and S2 multimers (8μg/ml) and observed under the microscope twice daily for five days.Photographs (100× normal or 200+ phase microscopy) were taken on days 1,2, 3, 4, and 5. The parameters observed, compared, and photographedincluded: 1) cell clumping that was similar to clumping in the T cellmitogen staphylococcal enterotoxin A (SEA) treated cultures (0.2 μg/ml),and 2) large cell formation, either a) dendritic type, i.e., branchedand/or elongated, or b) monocyte type, i.e., large oval. Observationsand photographs showed that qualitative and quantitative types ofmorphological changes occurred depending on the multimers used to treatthe cultures. The changes that occurred in the M1 treated cultures (8μg/ml) by day 2 are representative of the overall changes. Similarchanges occurred in the M2 treated cultures but were more pronouncedthan in the M1 treated cultures. These types of changes have occurred insix different human blood samples. In studies where the multimers weretitrated in cultures, significantly different responses occurred downto<1.0 μg/ml. Thus, the multimers are potent.

A media control showed definitely less clumping than the others and theclumps were less dense. The clumping results were routinely observed incultures treated with SEA. Treating cultures with the M2 stock thatcontained all the multimers resulted in the number of clumps being fewerthan in the other treated cultures but more dense than those treatedwith the multimers eluted from the upper band (1) and next lower band(2). Cultures treated with band 1 contained numerous large clumps, whilethose treated with band 2 also contained larger clumps but were fewer innumber. The clumps in both of these cultures were much larger than thoseobserved in the SEA treated cultures. Numerous compact clumps wereformed in cultures with bands 3 and 4. Band 3 caused larger clumpformation while band 4 caused more numerous clumps to form. Induction orenhancement of cell adhesion factors, e.g., CD2, LFA-1, VLA, ICAM-1etc., could be heavily involved in the observed clumping phenomena. Whenthe cultures were tested for presence of IL-2 by ELISA, only the SEAstimulated cultures were positive; thus, the multimers are not mitogenicfor naive T lymphocytes and cannot be considered as superantigens.Superantigens are known to induce apoptosis or toxicity and thereforewould be considered as harmful components of a vaccine or othercompounds to be used in vivo.

Cultures treated with the stock S1 and S2 monomers showed some minimalclumping. However, they did induce significant changes in the morphologyof some cells. Both multimers caused the formation of numerous, large,round cells; and long, sometimes bulging cells, often with noticeablebranching, while cultures treated with SEA lacked these morphologies.These cells resemble activated monocytes and dendritic cells, thus, theyappear to be important in antigen presentation. Interestingly, culturesstimulated with stock S1 and S2 produced low levels of interferon gamma,an important immunomodulatory lymphokine. Since interleukin-2 (IL-2) wasnot detected in these cultures, the interferon gamma (IFN) was mQstlikely not induced by IL-2; thus, its production resembles that of theearly IFN gamma response that is thought to be important inup-regulating the immune response.

Together these findings show that there are significant differences inthe biological activities of the stock multimers and the different sizesof multimers that can be isolated from them.

EXAMPLE 5 INDUCTION OF CYTOKINES IN NAIVE HUMAN BLOOD LEUKOCYTES

Because of the morphological changes observed in human leukocytecultures treated with various multimers and the overall immunoactivityof multimers, their ability to induce cytokines associated withimmunomodulation was investigated. The results are presented herein.

Briefly, enriched blood leukocyte cultures from one or two subjects wereprepared as described in Example 4. Cultures were then incubated forfour days in the presence of the indicated peptides and multimers (8μg/ml) shown in FIG. 9 and FIG. 10. Following incubation, thesupernatants were assayed for IL-1β, TNF-α, IFN-γ, and IL-10 by theELISA method. IL-1β, TNF-α, and IFN-γ have been shown to be importantup-regulators of immunoactivity and IL-10 is an importantdown-regulator.

The results (FIG. 9, FIG. 10 and summary Table 5) indicate that theleukocytes from the two subjects reacted similarly for the production ofIl-1β and TNF-α, i.e., many of the same peptides induced similarresponses in both subjects when compared to the controls.

                  TABLE 5                                                         ______________________________________                                        Summary of Biological Activities of Multimers.sup.a                           High            Medium       Low to +/-                                       ______________________________________                                        Lymph node proliferative response in immunized mice.                          ICR mice                                                                              S2 stock    S1 stock     M2 stock                                             M2 band 2   M2 band 1                                                 BALB/c  S1 stock    S2 stock                                                  mice                                                                          Cytokine induction in naive human leukocytes.                                 IFNγ                       M2 stock and                                                                  mixtures of M1                                                                and M2 bands                                 IL-1β                                                                            S2 stock and                                                                              S1 stock     M1 band 4 and                                        S1 stock + S2                                                                             M1 stock and top 3                                                                         various linkers                                      stock.sup.b M1 bands                                                                      M2 RL                                                     TNF-α                                                                           S2 stock, M1 and                                                                          M1 band 1 + M2                                                                             M1 band 4 and                                        2 + M2 Band 2                                                                             band 1, M2 LL                                                                              various linkers                                      M2 right linker                                                                           + M2 RL, S1  M2 stock                                             M1 band 1   stock + S2                                                        M1 band 2   stock                                                     IL-10   M1 band 1   M1 bands 2 and                                                                             various M1, M2,                                      M1 band 3 + M2                                                                            3 M2 band 1  S1 and S2                                            band 3, M1 LL +                                                                           S1 stock + S2                                                                              linker                                               M2 RL, M1 RL +                                                                            stock        combinations                                         M2 LL, S1 stock                                                       Morphological changes in naive                                                leukocyte cultures: Cell clumping.                                            Larger and                                                                            M2 band 2   M2 band 1    M2 stock                                     fewer                                                                         Smaller and                                                                           M2 band 3   M2 band 4                                                 more                                                                          numerous                                                                      Macrophage                                                                            S1 stock                                                              and dendritic                                                                         S2 stock                                                              changes S1 stock + S2                                                                 stock                                                                 ______________________________________                                         .sup.a -- Relative activity of multimers compared to controls.                .sup.b -- All combinations of peptides were used as mixtures.            

Interestingly, some stock multimers and some, but not all, of themultimers isolated from gel bands isolated from those stocks wereactive. Thus there is specificity for the activity of the variousmultimers isolatable by PAGE performed on active stock preparations.Also, as expected, good inducers of IL-1β were also good inducers ofTNF-α, since IL-1β induces synthesis of TNF-α. Overall, the studiesindicate that stock preparations of multimers, multimeric speciesisolatable from stocks, or various combinations of these can be used toobtain various degrees of induction activity. This may be very importantfor optimizing responses at local systemic or oral sites of vaccineadministration. In this regard, although this study was performed onhuman leukocytes, experiments in mice resulted in excellent lymph nodeproliferative responses (Table 1, FIG. 7 and FIG. 8). In addition, someof the linker sequences are active by themselves and thus may bevaluable as adjuvants, e.g., the M2 right linker.

FIG. 11 shows a representative experiment of the IFN-γ response, asmeasured by ELISA. Inducers of IFN-γ, were mostly limited to theisolated M1 bands, M2 stock and mixtures of M1 and M2 bands. No IL-2 wasdetectable in the supernatants suggesting that the induction of IFN-γwas IL-2 independent and thus could be related to the early IFN-γresponse that may be immunoenhancing. Since all the other multimerstested were negative, the data again indicate the specificity of certainmultimers for a biological activity.

FIG. 12 shows the IL10 response of naive human leukocytes treated withthe various peptide multimers and stocks. The data indicate that thehigher molecular weight multimers of M1 were better than the stock M1.Interestingly, the combination of lower sized M1 band 3 and M2 band 3multimers was more active than any other combination. Thus, combinationsof multimers may have activity that is different from the activities ofthe individual multimers that make them up. Combinations of the M1 andM2 linkers were also active and may be useful in suppressing immuneresponses. The S2 stock was also active and was also used tosuccessfully immunize ICR but not BALB/c mice (FIG. 7 and FIG. 8). Thisability to modulate human cytokines in leukocyte cultures provides asource of natural cytokines for use in humans or animals. The procedurehere is the same as described in Example 7 below, except that humanreagents are used.

EXAMPLE 6 USE OF MULTIMERS TO DETECT HIV ANTIBODIES IN SERA

The present inventors have been able to assay antibody in known HIVpositive sera in an ELISA, using the multimers containing the HIVepitope to bind the antibody (FIG. 13, 14 and 15). The titerwas>1/64000/0.1 ml using an EIA assay with S1 peptide bound to wells.The bound S2 peptide was less efficient (FIG. 13 and 15) and depended onthe amount of peptide bound to the wells. The epitope HP-6 alone did notbind antibody or normal serum IgG immunoglobulin. The ability to assayantibody to a single epitope has important implications for thespecificity and usefulness of the anti-HIV antibody test.

The binding activity in the serum was measured by a routine sandwichmethod ELISA in which the indicated, 0.5 to 20 μg/well, of peptidedissolved in carbonate buffer was adsorbed overnight to microtiterwells. The following morning the wells were blocked with 0.1 percentbovine serum albumin. Then a 1:20 dilution of known positive patientserum or known negative control serum was added to each well for twohours followed by washing three times with PBS TWEEN-20. Affinitypurified goat antibody to human IgG, conjugated to alkaline phosphatase(BIO-RAD) was then added followed by washing three times with PBSTWEEN-20. Phosphatase substrate was added and the color indicatorallowed to develop for 30 minutes. The optical density (OD) was thenread at 405 nm in a Molecular Devices Microplate Reader. The ODmeasurements resulting from binding of serum IgG to different quantitiesof peptide are shown in FIG. 13 and FIG. 14.

The present inventors envision the construction of a library of peptidesfor screening the responses of HIV patients for antibody and cellularresponses to various antigens. Information gained in this way fromsurvivors or long term survivors could be used to construct a vaccinethat induced similar responses. In addition, it is known that theantigens in the multimers are being expressed in proper form for avaccine and have much higher affinity for specific antibody than theantigen alone. This finding also strongly supports the data indicatingthe lymph node proliferative responses in mice were specific for the HIVepitope interlinked in the multimers.

The fact that the binding is specific is indicated from the data inTable 6. This table shows the results of testing known positive (11patients) and negative (15 subjects) serum samples from HIV positive andHIV negative patients. The data indicate that 10/11 positives wereinterpreted correctly, including #283-148 which was a +/- test in theAbbott EIA and 14/15 of the negative samples were interpreted correctlyusing an O.D. of 500 as the cut-off point.

Although the S2 peptide does not appear to be quite as efficient inbinding antibody as the S1, the S2 peptides were used in these studiesto strengthen the case that the lymph node proliferation abilities of S2in ICR mice was specific for the HP-6 epitope and, often, the lesssensitive test is the most specific. Also, ICR mice are outbred, as arehumans. Data in FIG. 14 suggest that the HP-6 epitope in S1 isresponsible for binding the antibody present in this known positiveserum obtained from the NIH. Thus, the epitopes in the S1 and S2peptides are present on the surface of the multimers in proper form.Therefore, ELISA assays could be developed using them that would becheaper to produce than those presently used. Also, ELISA assays couldbe used to detect antibodies to various epitopes of the virus and mutantvariations of the virus that would have important prognostic value andvalue for development of effective vaccines for many viruses. Also,ligands important in many biological activities could be presented inthe multimers for activating or blocking purposes.

                  TABLE 6                                                         ______________________________________                                        Results of ELISA Test for Antibody in HIV                                     Positive or Negative Serum Using S2 Peptide (5 μg or 1 μg/well)         Patient and            Result of    Known                                     Serum Sample No.                                                                          Absorbance Present Invention                                                                          Result.sup.a                              ______________________________________                                                  5 μg/well                                                        5           0.071      -            -                                         3           0.101      -            -                                         8           0.164      -            -                                         9           0.169      -            -                                         4           0.178      -            -                                         1           0.186      -            -                                         10          0.218      -            -                                         300-1598    0.243      .sup. -.sup.b                                                                              +                                         2           0.272      -            -                                         300-1665    0.304      -            -                                         6           0.336      -            -                                         307-1850    0.412      -            -                                         7           0.445      -            -                                         299-2151    0.446      -            -                                         8025        0.501      +/-          -                                         300-1856    0.530      +            +                                         300-1593    0.539      +            +                                         283-1448    0.588      +            +/-.sup.c                                 306-1669    0.670      +            +                                         307-1758    0.708      +            +                                         306-2872    1.061      +            +                                         300-1773    1.265      +            +                                                   1 μg/well                                                        264-1699    0.169      -            -                                         300-1861    0.363      +            +                                         306-2046    0.386      +            +                                         304-2945    0.506      +            +                                         NIH.sup.d   0.760      +            +                                         ______________________________________                                         .sup.a -- Abbott Laboratories EIA                                             .sup.b -- Possible no antibody to HP6 epitope                                 .sup.c -- Positive by fluorescence test                                       .sup.d -- Known positive serum from National Institute of Health         

Since the M peptides do not bind antibody in HIV positive serum, itappears that the antigens are not accurately portrayed on the surface ofthe multimers. However, the data of Table 1 demonstrate that, if miceare immunized with the M2 band 2 multimer, a specific lymph nodeproliferative response to HP-6 is elicited. Thus, it would appear thatthe antigens become accurately presented on the surface of antigenpresenting cells after intracellular processing of the non-covalentlybound multimers. Unlinked peptides are normally presented directly onthe surface of the cell and for this reason are often less immunogenic.This type of presentation of peptide antigen is useful for intracellularmodulation of the immune response. Practically, this means that antigensthat are not presented properly by a defective immune system may, infact, be sandwiched with linkers and presented properly. In addition,although the M1 and M2 monomers did not bind HIV antibody (probablybecause the peptides are not situated in the proper position on themultimers), they and their linkers' abilities to induce IL-1β, TNF-α,IFN-gamma, and IL-10 are still important, as well as their ability toimmunize mice to the extent that their lymph node cells respond tostimulation by the HIV epitope, HP-6 (Table 1). Again, this latter factprobably results from the way the non-covalently bound multimers areprocessed by antigen presenting cells.

EXAMPLE 7 INDUCTION OF ANTIBODIES TO HIV EPITOPE HP-6 FOLLOWINGIMMUNIZATION WITH MULTIMERIC PEPTIDES

ELISA Assay for Antibody:

The antibody in the serum was measured by a routine sandwich methodELISA assay in which 2.0 μg/well of peptide dissolved in carbonatebuffer was adsorbed overnight to microtiter wells. The following morningthe wells were blocked with 0.1 percent bovine serum albumin. Then a1:20 dilution of serum was added to each well for two hours followed bywashing three times with PBS Tween-20. Polyclonal antibody to mouse IgM,IgG and IgA, or affinity purified antibody to IgG, conjugated toalkaline phosphatase (BIO-RAD) was then added followed by washing threetimes with PBS Tween-20. Phosphatase substrate was added and the colorindicator allowed to develop for 30 minutes. The optical density (OD)was then read at 405 nm in a Molecular Devices Microplate Reader. The ODmeasurements are shown in Tables 7, 8, 9 and 10. The results, ODreadings at least four fold greater than controls and OD readingsgreater than 0.125 and statistical significance, when possible todetermine, are underlined readings with asterisks.

Results:

Groups of ICR mice, three (A, B, C)/group, were injected subcutaneouslyat the base of the tail with the indicated multimeric peptidesemulsified in Freund's complete (FCA) or incomplete adjuvant (FIA).Blood was collected and pooled at three weeks post-immunization andtested (see ELISA assay above) singly to screen for early development ofIgM, IgA and/or IgG antibody to multimers (Table 7).

                                      TABLE 7                                     __________________________________________________________________________    Levels of Antibodies to Multimers and HIV Epitope HP-6 in                     Individual ICR Mice 3 and 5 Weeks after Immunization                                 FCA               IFA                                                  ANTISERUM/                                                                           3 WKS       5 WKS 3 WKS       5 WKS                                    ANTIGEN                                                                              CTRL                                                                              POOLED                                                                             A  B  C  CTRL                                                                              POOLED                                                                             A  B  C                                     __________________________________________________________________________    M1/M1  .071                                                                              .173 .098                                                                             .133                                                                             .173                                                                             .029                                                                              .094 .092                                                                             .070                                                                             .077                                  M2/M2  .013                                                                              .049 .124                                                                             .021                                                                             .030                                                                             .000                                                                              .080 .032                                                                             .051                                                                             .021                                  S1/S1  .065                                                                              .482 .525                                                                             .081                                                                             .204                                                                             .033                                                                              .223 .509                                                                             .244                                                                             .026                                  S2/S2  .100                                                                              .112 .118                                                                             .091                                                                             .081                                                                             .040                                                                              .070 .062                                                                             .058                                                                             .088                                  M2.B1/M2.B1                                                                          --  .018 -- .002                                                                             .003                                                                             .025                                                                              .029 .029                                                                             .035                                                                             .066                                  M2.B2/M2.B2                                                                          .004                                                                              .047 .020                                                                             .012                                                                             .034                                                                             .025                                                                              .044 .044                                                                             .028                                                                             .035                                  M2.B3/M2.B3                                                                          .002                                                                              --   .018                                                                             .007                                                                             .012                                                                             .010                                                                              .027 .014                                                                             .016                                                                             .020                                  M2.B1/M2                                                                             .002                                                                              .038 .006                                                                             .027                                                                             .036                                                                             -.010                                                                             .064 .000                                                                             .001                                                                             .066                                  M2.B2/M2                                                                             .009                                                                              .078 .040                                                                             .019                                                                             .028                                                                             .001                                                                              .073 .011                                                                             .003                                                                             .037                                  M2.B3/M2                                                                             .005                                                                              --   .032                                                                             .013                                                                             .011                                                                             .000                                                                              .021 .000                                                                             .000                                                                             .012                                  FCA/FCA                                                                              .001                                                                              .006 .002                                                                             .001                                                                             -.010                                                   IFA/IFA                  .002                                                                              .000 -.001                                                                            .003                                                                             .010                                  M1/HP.6                                                                              .010                                                                              .234 .086                                                                             .147                                                                             .222                                                                             .010                                                                              .131 .103                                                                             .035                                                                             .036                                  M2/HP.6                                                                              .005                                                                              .162 192                                                                              .116                                                                             .074                                                                             --  .117 .015                                                                             .046                                                                             .030                                  S1/HP.6                                                                              .014                                                                              .333 .151                                                                             .027                                                                             .257                                                                             .003                                                                              .156 .055                                                                             .225                                                                             .005                                  S2/HP.6                                                                              .008                                                                              .085 .201                                                                             .110                                                                             .038                                                                             .005                                                                              .115 .085                                                                             .071                                                                             .124                                  M1/S2.20                                                                             .009                                                                              .163 .062                                                                             .109                                                                             .172                                                                             .008                                                                              .026 .009                                                                             .010                                                                             .016                                  M2/S2.20                                                                             .003                                                                              .114 .139                                                                             .073                                                                             .061                                                                             .001                                                                              .025 .002                                                                             .013                                                                             .009                                  S1/S2.20                                                                             .008                                                                              .242 .092                                                                             .020                                                                             .161                                                                             .007                                                                              .024 .006                                                                             .023                                                                             .002                                  S2/S2.20                                                                             .018                                                                              .071 .094                                                                             .073                                                                             .025                                                                             .004                                                                              .024 .023                                                                             .022                                                                             .048                                  M1/S2.22                                                                             .004                                                                              .061 .042                                                                             .056                                                                             .065                                                                             .005                                                                              .010 .017                                                                             .021                                                                             .027                                  M2/S2.22                                                                             .005                                                                              .061 .075                                                                             .044                                                                             .038                                                                             .003                                                                              .016 .003                                                                             .017                                                                             .010                                  S1/S2.22                                                                             .010                                                                              .152 .181                                                                             .020                                                                             .185                                                                             .001                                                                              .017 .005                                                                             .025                                                                             --                                    S2/S2.22                                                                             .003                                                                              .036 .058                                                                             .057                                                                             .030                                                                             --  .012 .023                                                                             .031                                                                             .048                                  M2.B1/HP.6                                                                           .014                                                                              .137 .020                                                                             .020                                                                             .030                                                                             .005                                                                              .093 .009                                                                             .001                                                                             .027                                  M2.B2/HP.6                                                                           .020                                                                              .165 .090                                                                             .141                                                                             .071                                                                             .003                                                                              .141 .017                                                                             .012                                                                             .064                                  M2.B3/HP.6                                                                           .013                                                                              .008 .097                                                                             .045                                                                             .015                                                                             .007                                                                              .025 .006                                                                             .007                                                                             .009                                  FCA/HP.6                                                                             0.19                                                                              --   .032                                                                             .081                                                                             .004                                                    IFA/HP.6                 .016                                                                              .034 .023                                                                             .028                                     M2.B1/S2.20                                                                          .007                                                                              .099 .027                                                                             .021                                                                             .038                                                                             .066                                                                              .025 .006                                                                             .003                                                                             .016                                  M2.B2/S2.20                                                                          .009                                                                              .145 .070                                                                             .086                                                                             .041                                                                             .008                                                                              .029 .019                                                                             .005                                                                             .011                                  M2.B3/S2.20                                                                          .009                                                                              .000 .069                                                                             .025                                                                             .000                                                                             .006                                                                              .013 .004                                                                             .005                                                                             .004                                  FCA/S2.20                                                                            .010                                                                              --   .020                                                                             .039                                                                             .000                                                    IFA/S2 .20               .014                                                                              .016 .014                                                                             .006                                     M2.B1/S2.22                                                                          .004                                                                              .062 .011                                                                             .024                                                                             .039                                                                             .010                                                                              .007 .000                                                                             .001                                                                             .013                                  M2.B2/S2.22                                                                          .011                                                                              .103 .055                                                                             .057                                                                             .074                                                                             .000                                                                              .020 .016                                                                             .002                                                                             .009                                  M2.B3/S2.22                                                                          .018                                                                              .016 .039                                                                             .020                                                                             .007                                                                             .001                                                                              .005 .000                                                                             .001                                                                             .001                                  FCA/S2.22                                                                            .008                                                                              --   .027                                                                             .035                                                                             .002                                                    IFA/S2.22                .006                                                                              .009 .011                                                                             .001                                     __________________________________________________________________________

Serum collected at five weeks post immunization from individual mice wasalso tested singly for screening purposes (Table 7). Table 8 shows theresults and significance (Student's t test) of selected individual serumsamples from the mice in Table 7 when measured in duplicate forstatistical purposes and when sufficient sample was available.

                                      TABLE 8                                     __________________________________________________________________________    Levels of Antibodies to Multimers in Individual ICR Mice 3 and 5 Weeks        After Immunization                                                            FCA                           FIA                                             Antiserum/  3 WEEKS                                                                            5 WKS              3 WEEKS                                                                            5 WKS                                Antigen                                                                             CONTROL                                                                             POOLED                                                                             A   B    C   CONTROL                                                                             POOLED                                                                             A   B   C                            __________________________________________________________________________    S/M1  0.007 0.218**                                                                            0.100                                                                             0.008                                                                              0.076                                                                             0.002 0.269*** 0.130*                                                                            0.068                        S2/M1 <0.0  0.035                                                                              0.037                                                                             0.042                                                                              0.044     0.502**                                   M1/M1 0     0.512**                                                                            0.687**                                                                           0.353**                                                                            0.107*                                                                            0.00       0.443*                                                                            0.353*                                                                            0.457*                       M2/M1 0.010      0.028                                                                             0.043                                                                              0.194                                               M2.B2/M1                                                                            <0.0  0.100*                                                                             0.066                                                                             0.043                                                                              0.021                                               M2.B3/M1                                                                            05         0.004    0.036                                                     0.0                                                                           0.003                                                                   S1/M2 0.007 0.218**                                                                            0.120                                                                             0.023                                                                              0.038                                                                             0.00  0.097    0.097                                                                             0.025                        S2/M2 0.00  0.026                                                                              0.017                                                                             0.041                                                                              0.066                                               M1/M2 0.011 0.137*                                                                             0.084                                                                             0.059                                                                              0.038                                                                             0.005 0.071                                                                              0.064                                                                             0.010                                                                             0.009                        M2/M2 0.00       0.015                                                                             0.026                                                                              0.181*                                              M2.B2/M2                                                                            0.003 0.069                                                                              0.050                                                                             0.022                                                                              0.009                                               M2.B3/M2                                                                            0.000      0.003    0.029                                               S1/S1 <0.0  0.663**                                                                            0.090                                                                             <0.0     0.033 0.282**  0.084                                                                             0.839**                      S2/S1 0.012 0.005                                                                              <0.0                                                                              0.041                                                                              0.890**                                             M1/S1 <0.0  0.097                                                                              0.075                                                                             0.293**                                                                            <0.0                                                                              0.006 0.525*                                                                             0.285**                                                                           0.144                                                                             0.092                        M2/S1 0.014      <0.0                                                                              <0.0 <0.0                                                M2.B2/S1                                                                            <0.0  0.035                                                                              0.047                                                                             0.037                                                                              0.039                                               M2.B3/S1                                                                            <0.0  0.069                                                                              <0.0     0.025                                                                         <0.0                                                S1/S2 0.022 0.197**                                                                            0.134*                                                                            0.029                                                                              0.096                                                                             <0.0  0.171**  0.103**                                                                           0.318**                      S2/S2 0.001 0.047                                                                              0.037    0.035                                               M1/S2 0.057 0.334*                                                                             0.407*   0.040                                                                             0.008 0.446*                                                                             0.202**                                                                           0.114**                                                                           0.045                        M2/S2 0.018      0.046                                                                             0.406***                                                                           0.216**                                             M2.B2S21                                                                            0.012 0.112**                                                                            0.072                                                                             0.208*                                                                             0.004                                               M2.B3/S2                                                                            0.029      0.070                                                                             0.035                                                                              0.055                                                                    0.055                                                    __________________________________________________________________________     *P ≦ 0.005                                                             *P ≦ 0.005                                                             ***P = 0.1 to 0.049                                                      

Table 9 shows the results and significance of duplicate measurements ofthe antibody response to multimers and linkers five weeks after thesecond immunization with the indicated multimers.

                                      TABLE 9                                     __________________________________________________________________________    Levels of Antibodies to Multimers and HIV Epitope HP-6 Linkers and in         Individual                                                                    ICR Mice 5 Weeks After Second Immunization                                    Antiserum/                                                                          FCA            FIA                                                      Antigen                                                                             A    B    C    A     B    C                                             __________________________________________________________________________    S1/M1 0.075                                                                              0.133***                                                                           0.303**                                                                            0.166*                                                                              0.077                                              S2/M1 0.047                                                                              0.114                                                                              0.067                                                                              0.062 0.090                                                                              0.108**                                       M1/M1 0.307*                                                                             0.074                                                                              0.354*                                                                             0.250***                                                                            0.323***                                                                           0.335***                                      M2/M1 0.257**                                                                            0.022                                                                              0.083                                                                              0.128**                                                                             0.243*                                                                             --                                            M2.B1/M1                                                                            0.053                                                                              0.037                                                                              0.013                                                                              0.086 --   0.168**                                       M2.B2/M1                                                                            0.108                                                                              0.098                                                                              0.269*                                                                             0.061 0.023                                                                              0.014                                         M2.B3/M1                                                                            0.060                                                                              0.02 0.072                                                                              0.001 0.012                                                                              0.021                                         FCA/M1                                                                              0.085                                                                              0.078                                                                              --   0.067 0.047                                                                              --                                            S1/M2 0.034                                                                              0.078                                                                              0.217**                                                                            0.085 0.047                                                                              --                                            S2/M2 0.022                                                                              0.051                                                                              0.042                                                                              0.057 0.067                                                                              0.110**                                       M1/M2 0.081                                                                              0.084                                                                              0.087                                                                              0.034 0.043                                                                              0.117**                                       M2/M2 0.148*                                                                             0.017                                                                              0.047                                                                              0.076 0.167*                                                                             --                                            M2.B1/M2                                                                            0.020                                                                              0.025                                                                              0.026                                                                              0.103*                                                                              0.003                                                                              0.076                                         M2.B2/M2                                                                            0.069                                                                              0.066                                                                              0.100                                                                              0.022 0.023                                                                              0.021                                         M2.B3/M2                                                                            0.038                                                                              0.005                                                                              0.012                                                                              0.008 0.014                                                                              0.024                                         FCA/M2                                                                              0.072                                                                              0.059                                                                              --   0.057 0.028                                                                              --                                            S1/S1 0.231**                                                                            0.390***                                                                           0.752**                                                                            0.648**                                                                             0.664*                                                                             --                                            S2/S1 0.017                                                                              0.058                                                                              0.012                                                                              0.047 0.095                                                                              0.080                                         M1S1  0.255**                                                                            0.075                                                                              0.089                                                                              0.034 0.223**                                                                            0.200*                                        M2/S1 0.108*                                                                             0.024                                                                              0.016                                                                              0.075 0.153**                                                                            --                                            M2.B1/S1                                                                            0.035                                                                              0.022                                                                              0.023                                                                              0.041 0.003                                                                              0.094                                         M2.B2/S1                                                                            0.089                                                                              0.046                                                                              0.126*                                                                             0.015 0.031                                                                              0.026                                         M2B3/S1                                                                             0.053                                                                              0.008                                                                              0.109                                                                              0.003 0.012                                                                              0.0                                           FCA/S1                                                                              0.039                                                                              0.049                                                                              --   0.053 0.029                                                                              --                                            S1/S2 0.053                                                                              0.067                                                                              0.140**                                                                            0.063 0.159*                                                                             --                                            S2/S2 0.040                                                                              0.084                                                                              0.076                                                                              0.027 0.069                                                                              0.054                                         M1/S2 0.082                                                                              0.044                                                                              0.105*                                                                             0.036 0.066                                                                              0.201*                                        M2/S2 0.214**                                                                            0.019                                                                              0.010                                                                              0.107*                                                                              0.224*                                                                             --                                            M2/B1/S2                                                                            0.045                                                                              0.027                                                                              0.057                                                                              0.076 0.003                                                                              0.069                                         M2.B2/S2                                                                            0.164*                                                                             0.119                                                                              0.177***                                                                           0.013 0.015                                                                              0.016                                         M2.B3/S2                                                                            0.050                                                                              0.036                                                                              0.065                                                                              0.019 0.012                                                                              0.041                                         FCA/S2                                                                              0.024                                                                              0.116     0.055 0.075                                                                              --                                            M1/HP.6                                                                             0.083                                                                              0.131                                                                              0.162*                                                                             0.028 0.042                                                                              0.070                                         M2/HP.6                                                                             0.126*                                                                             0.034                                                                              0.121*                                                                             0.118***                                                                            0.127*                                                                             --                                            S1/HP.6                                                                             0.033                                                                              0.105***                                                                           0.388**                                                                            0.194**                                                                             0.033                                                                              --                                            S2/HP.6                                                                             0.044                                                                              0.084                                                                              0.060                                                                              0.064*                                                                              0.120**                                                                            0.147**                                       M2.B1/HP.6                                                                          0.019                                                                              0.044                                                                              0.010                                                                              0.006 0.004                                                                              0.056                                         M2.B2/HP.6                                                                          0.088                                                                              0.097*                                                                             0.110*                                                                             0.024 0.032                                                                              0.029                                         M2.B3/HP.6                                                                          0.028                                                                              0.013                                                                              0.025                                                                              0.017 0.001                                                                              0.023                                         FCA/HP.6                                                                            0.051                                                                              0.051                                                                              0.051                                                         FIA/HP.6             0.035 0.035                                                                              0.035                                         S1.S1.LL                                                                            0.013                                                                              0.014                                                                              0.117**                                                                            0.035 0.011                                                                              --                                            S2.S1.LL                                                                            0.008                                                                              0.013                                                                              0.018                                                                              0.030 0.069                                                                              0.050                                         S1/S2.LL                                                                            0.016                                                                              0.020                                                                              0.140**                                                                            0.035 0.010                                                                              --                                            S2/S2.LL                                                                            0.012                                                                              0.014                                                                              0.013                                                                              0.024 0.044                                                                              0.045                                         FCA/S1.LL                                                                           0.017                                                                              0.034                                                                              --   0.025 0.008                                                                              --                                            FIA/S1.LL                                                                     S1/S1.RL                                                                            0.021                                                                              0.075                                                                              0.198*                                                                             0.071 0.020                                                                              --                                            S2/S1.RL                                                                            0.006                                                                              0.018                                                                              0.026                                                                              0.040 0.069                                                                              0.065                                         S1/S2.RL                                                                            0.004                                                                              0.011                                                                              0.155**                                                                            0.022 0.003                                                                              --                                            S2/S2.4L                                                                            0.005                                                                              0.007                                                                              0.005                                                                              0.018 0.034                                                                              0.057                                         FCA/S1.RL                                                                           0.050                                                                              0.025                                                                              --              --                                            FIA/S1.RL            0.031 0.009                                              FCA/S2.RL                                                                           0.011                                                                              0.020                                                                              --              --                                            FIA/S2.RL            0.025 0.005                                              __________________________________________________________________________     *P ≦ 0.05                                                              **P ≦ 0.005                                                            ***P = 0.1 to 0.049                                                      

Table 10 shows the results and significance of the IgG response ofindividual mice to the multimers five weeks after the secondimmunization, when sufficient sample was available.

                                      TABLE 10                                    __________________________________________________________________________    Levels of IgG antibody to peptides of individual ICR mice 5 weeks after       second                                                                        Antiserum/Peptide                                                                      FCA            FIA                                                   Antigen  A    B    C    A    B    C                                           __________________________________________________________________________    S1/S1    1.190**                                                                            2.163***                                                                           2.148***                                                                           2.30***                                                                            2.325***                                                                           --                                          S2/S1    0.060                                                                              0.140***                                                                           0.124***                                                                           0.170***                                                                           0.537***                                                                           0.202**                                     FCA/S1   0.1775                                                                             0.133                                                                              --                                                         FIA/S1                  0.176                                                                              0.098                                                                              --                                          Control/S1                                                                             0.013          0.013                                                 S1/S2    0.123                                                                              0.390*                                                                             1.00***                                                                            0.525***                                                                           0.768***                                                                           --                                          S2/S2    0.82 0.163*                                                                             0.132                                                                              0.091                                                                              0.321**                                                                            0.134                                       FCA/S2   0.093                                                                              0.099                                                           FIA/S2                  0.170                                                                              0.116                                            Control/S2                                                                             0.014          0.014                                                 M1/M1    1.518***                                                                           0.115                                                                              1.637***                                                                           1.476***                                                                           1.576***                                                                           1.592***                                    M2/M1    0.832***                                                                           0.045                                                                              0.124***                                                                           0.583***                                                                           1.049***                                                                           --                                          FCA or FIA/M1                                                                          0.060                                                                              0.034     0.071                                                                              0.028                                            Control/M1                                                                             0.007          0.007                                                 M1/M2    0.068                                                                              0.01 0.124                                                                              0.009                                                                              0.020                                                                              0.189*                                      M2/M2    0.393*                                                                             0.021                                                                              0.02 0.102                                                                              0.389                                                                              --                                          FCA or FIA/MS                                                                          0.040                                                                              0.015                                                                              --   0.046                                                                              0.007                                                                              --                                          Control/M2                                                                             0.002          0.002                                                 __________________________________________________________________________     *P ≦ 0.05                                                              **P ≦ 0.01                                                             ***P ≦ 0.001                                                           doubly underlined values indicate where crossreactivity occurred.        

The data in Tables 7 and 8 show that antibodies to multimeric peptidesS1 and M1 were induced against S1 and M1, respectively, in two out ofthree mice in the presence of FCA or FIA. More importantly all the stockpeptides induced antibody to the HIV HP-6 peptide in one to two mice pergroup of three, indicating that the multimer could induce specificantibody to the HIV epitope sandwiched between the linkers. Multimers S1and M1 worked most effectively in this study. Antibodies to S1 alsoreacted with the truncated S20 and S22 peptides, further indicatingspecificity. Antibodies to M1 also reacted with HP-6, but to a lesserextent. Antibodies to HP-6 were also induced by M2 band 2, the samemultimer that induced a significant proliferative response in these mice(Table 1). Thus, this result supports the specificity of theproliferative response for HP-6.

Table 9 shows the potential IgM, IgA and IgG antibody responses to themultimers, HP-6 and linkers five weeks after the second immunization. Itwas observed that all the mice tested produced significant amounts ofantibody to the S1 and S2 peptides. S1 also induced antibodies in someof the mice immunized with M1, S2 or M2 peptides. The only commonmodality in these peptides with the S1 peptide is the HP-6 epitope, thusthe data suggests that the antibodies detected reacted to the HP-6epitope. This is supported by the fact that antibodies to S1 alsoreacted directly with HP-6 in three out of four mice although one was alow responder. The M2 peptide also induced low levels of antibody toHP-6 in three out of four mice. Sero conversions also occurred followingthe second immunization with the S1 and M1 multimeric peptides.

Since the responses to the polyclonal pooled anti-IgM, IgA and IgGconjugated antibodies were lower than expected, the mouse serumsharvested from mice five weeks after the second immunization weretested, when sufficient amounts were available, in an ELISA usingconjugated affinity purified anti-IgG. Table 10 shows the IgG antibodyresponse of individual mice to the indicated multimer. It can be notedthat many of the antibody responses were very high, e.g., greater than1.0, and some were greater than 2, when the S1 peptide was used asimmunogen in the presence of FCA or FIA. This indicates that significantamounts of IgG antibody were induced in mice, even when the peptideswere given in Freund's incomplete adjuvant The fact that the antibodiescross-reacted with peptides with different linkers, but the same HP-6sequence (doubly underlined values), shows that the antibodies detectedare directed against the HP-6 moiety. The ability to induce high levelsof IgG antibodies also indicates that the multimers are able to inducememory cells. Thus the multimer-induced immune response is long term.

Tables 11 and 12 show summaries of the proportion of mice mounting anantibody response to individual multimers inoculated in Freund'scomplete (FCA) or incomplete adjuvant (FIA) five weeks after oneimmunization (Table 10) and five weeks after a second immunization(Table 11).

                                      TABLE 11                                    __________________________________________________________________________    Summary of Proportion of ICR mice with antibody to peptides and linkers       five weeks                                                                    after immunization with peptides.                                             S1        S2    M1    M2    M2 B2 M2 B3                                       Peptide                                                                           FCA.sup.a                                                                        FIA.sup.b                                                                        FCA                                                                              FIA                                                                              FCA                                                                              FIA                                                                              FCA                                                                              FIA                                                                              FCA                                                                              FIA                                                                              FCA                                                                              FIA                                      __________________________________________________________________________    S1  2/3                                                                              2/3                                                                              0/3                                                                              0/0                                                                              1/3                                                                              2/3                                                                              0/3                                                                              ND 0/3                                                                              ND 0/2                                                                              ND                                       HP-6                                                                              2/3                                                                              1/3                                                                              2/3                                                                              0/3                                                                              2/3                                                                              0/3                                                                              1/3                                                                              0/3                                                                              1/3                                                                              0/3                                                                              0/3                                                                              0/3                                      S2 20                                                                             1/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              1/3                                                                              0/3                                                                              1/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                      S2 22                                                                             2/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                      S1 LL                                                                             0/0                                                                              0/1                                                                              ND ND                                                               S1 RL                                                                             0/0                                                                              0/1                                                                              ND ND                                                               S2  1/3                                                                              1/2                                                                              0/3                                                                              ND 2/3                                                                              2/3                                                                              1/3                                                                              ND 0/3                                                                              ND 0/2                                                                              ND                                       S2 LL                                                                             0/0                                                                              0/2                                                                              ND ND                                                               S2 RL                                                                             0/0                                                                              0/2                                                                              ND ND                                                               M1  0/3                                                                              1/2                                                                              0/3                                                                              ND 2/3                                                                              3/3                                                                              1/3                                                                              ND 0/3                                                                              ND 0/2                                                                              ND                                       M1 LL                                                                             -- -- -- -- 0/1                                                                              1/3                                                                              0/0                                                                              0/0                                                                              0/0                                                                              0/0                                                                              -- --                                       M1 RL                                                                             -- -- -- -- 0/1                                                                              0/2                                                                              0/1                                                                              0/3                                                                              0/0                                                                              0/0                                                                              -- --                                       M2  0/3                                                                              0/2                                                                              0/3                                                                              ND 0/3                                                                              0/3                                                                              1/3                                                                              ND 0/3                                                                              ND 0/2                                                                              ND                                       M2 LL                                                                             -- -- -- -- 0/0                                                                              1/2                                                                              0/0                                                                              0/0                                                                              0/0                                                                              0/0                                                                              -- --                                       M2 RL                                                                             -- -- -- -- 0/1                                                                              0/1                                                                              1/1                                                                              1/1                                                                              1/1                                                                              ND -- --                                       M2-B2                       0/3                                                                              0/3                                            M2-B3                             0/3                                                                              0/3                                      __________________________________________________________________________     .sup.a = Freund's complete adjuvant                                           .sup.b = Freund's incomplete adjuvant                                    

                                      TABLE 12                                    __________________________________________________________________________    Summary of Proportion of ICR mice with antibody to peptides and linkers       five weeks                                                                    after second immunization with peptides.                                      S1        S2    M1    M2    M2B2  M2B3                                        Peptide                                                                           FCA.sup.a                                                                        FIA.sup.b                                                                        FCA                                                                              FIA                                                                              FCA                                                                              FIA                                                                              FCA                                                                              FIA                                                                              FCA                                                                              FIA                                                                              FCA                                                                              FIA                                      __________________________________________________________________________    S1  3/3                                                                              2/2                                                                              0/3                                                                              0/3                                                                              1/3                                                                              2/3                                                                              0/3                                                                              1/2                                                                              1/3                                                                              0/3                                                                              0/3                                                                              0/3                                      HP-6                                                                              2/3                                                                              1/2                                                                              0/3                                                                              2/3                                                                              2/3                                                                              0/3                                                                              1/3                                                                              1/2                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                      S1 LL                                                                             0/3                                                                              0/2                                                                              0/3                                                                              0/3                                                                              -- -- -- -- -- -- -- --                                       S1 RL                                                                             1/3                                                                              0/2                                                                              0/3                                                                              0/3                                                                              -- -- -- -- -- -- -- --                                       S2  1/3                                                                              1/2                                                                              0/3                                                                              0/3                                                                              0/3                                                                              1/3                                                                              1/3                                                                              2/2                                                                              2/3                                                                              0/3                                                                              0/3                                                                              0/3                                      S2 LL                                                                             1/3                                                                              0/2                                                                              0/3                                                                              0/3                                                                              -- -- -- -- -- -- -- --                                       S2 RL                                                                             1/3                                                                              0/2                                                                              0/3                                                                              0/3                                                                              -- -- -- -- -- -- -- --                                       M1  1/3                                                                              1/2                                                                              0/3                                                                              0/3                                                                              2/3                                                                              3/3                                                                              1/3                                                                              2/2                                                                              1/3                                                                              0/3                                                                              0/3                                                                              0/3                                      M1 LL     0/2                                                                              0/1                                                                              1/2                                                                              0/2                                                        M1 RL     0/2                                                                              0/1                                                                              0/2                                                                              0/2                                                        M2  1/3                                                                              0/2                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              1/3                                                                              1/3                                                                              0/3                                                                              0/3                                                                              0/3                                                                              0/3                                      M2 LL     0/2                                                                              0/1                                                                              1/2                                                                              0/2                                                        M2 RL     0/2                                                                              0/1                                                                              1/2                                                                              0/2                                                        __________________________________________________________________________     FCA = Freund's complete adjuvant                                              FIA = Freund's incomplete adjuvant                                       

It can be noted that the largest proportion of positive mice correspondto those mice inoculated with multimers that induced the highest levelsof antibody. Thus, certain multimers can induce high levels of antibodyin a high proportion of mice.

Overall the data illustrate that certain multimeric peptides are able toinduce high levels of antibody, including IgG antibody, in the absenceof complete adjuvant and with as few as two immunizations. The fact thatthe antibody cross-reacts with unrelated multimers containing the sameepitope as the immunogen indicates that the antibody detected wasspecific. This conclusion is supported by the ability of the antibodiesin some of the mice to react directly with the HP-6 epitope.

EXAMPLE 8 Generic construction of Linking Biologically Active Peptides

In general to ensure initial success the peptides should be fashionedsimilar to the S1 and S2 or M1 and M2 peptides described herein. But itis possible to use modifications, especially in regard to the length ofthe linkers, but the efficacy needs to be determined empirically. Anamphipathic epitope should be selected for the antigen of interest,however other linear sequences have not been ruled out. The linkersshould consist of 12 amino acid residues of alternating hydropathicity,but shorter linkers may serve as well. The linkers of biologicallyactive multimers are generally composed of alternating hydrophobic andhydrophilic amino acids as indicated in the S1 and S2 type peptides, butmore neutral spacing amino acids may be used that would allow forvarious kinds of folding resulting in different activities, as indicatedby the different activities of the multimers extracted from the gelbands for the M1 and M2 peptides. In fact, one may use naturallyoccurring amino acid sequences with similar alternating patterns to getlinked monomers with biological activity as indicated in the M1 peptide.Linkage can be checked by neutral polyacrylamide gel electrophoreses.Depending on the desired biological activity needed, bands may be mosteffectively eluted from gels using 10 to 100% DMSO followed by dilutionin Tris-HCl. It is likely that molecular sizing columns could also beused, because of the stability of the multimers. The stock multimers arealso very active for different biological functions and thus could bediluted and used neat as desired. The peptides may be synthesized, forexample, by the following method:

Peptides described herein were synthesized in the laboratory of Dr. H.M. Johnson on a Biosearch Model 9500AT automated peptide synthesizer(Millipore Corporation, Bedford, Mass.) using N.sup.α-((-fluoroenyl)methyloxycarbonyl chemistry (Chang et al.). Peptides werecleaved from the resins using trifluoroaceticacid/ethanedithiol/thioanisole/anisole at a ratio of 90/3/5/2. Thecleaved peptides were then extracted in ether and ethyl acetate andsubsequently dissolved in water and lyophilized. Reverse phase HPLCanalysis of the crude peptides revealed one major peak in each profile,hence additional purification was not warranted. Amino acid analyses ofthe peptides showed that the amino acid composition corresponded closelyto theoretical values and indicated a purity of greater than 90%. Table13 shows a typical amino acid analysis.

                  TABLE 13                                                        ______________________________________                                        Typical Amino Acid Analysis                                                   Amino Acid                                                                            Notes   Range (± 10%)                                                                         Rationale                                          ______________________________________                                        Cys-acid                                                                              1,2     1.00                                                          Asx             1.00                                                          Thr             1.00                                                          Ser             0.80       dehydration                                        Glx             1.00                                                          Gly             1.25       from degradation of other aa                       Ala     3       1.00                                                          Val             0.90       rearrangement                                      Met             0.7        S-alkylation, oxidation                            Ile             0.85       rearrangement                                      Leu     3       1.00                                                          Tyr     1       1.00                                                          Phe     2       1.00                                                          His     2       0.700-1.40 oxidation                                          Lys     2       1.00                                                          Trp     1,2     0.500-1.00 very unstable                                      Arg     2       1.00                                                          Pro              1.15-1.18                                                    ______________________________________                                         Notes:                                                                        1. Detection requires special hydrolysis                                      2. Frequently difficult to quantitate                                         3. Extremely stable                                                      

Table 14 shows amino acid analyses of inventive peptides synthesized.

                  TABLE 14                                                        ______________________________________                                        Peptide Amino Acid     Theoretical                                                                             Actual                                       ______________________________________                                        S1      Asx            4         4.3                                                  Ser            4         4.2                                                  Glx            5         5.1                                                  Ala            4         4.4                                                  Val            2         1.7                                                  Ile            7         6.3                                                  Leu            3         3.0                                                  Tyr            2         2.2                                                  His            2         1.4                                                  Lys            1         0.9                                                  Trp            1         --                                                   Arg            2         2.1                                          S2      Asx            5         4.6                                                  Ser            2         2.0                                                  Glx            4         4.5                                                  Gly            2         2.5                                                  Ala            2         2.1                                                  Val            2         2.0                                                  Ile            2         1.4                                                  Leu            6         6.3                                                  Tyr            3         3.2                                                  Phe            2         2.2                                                  His            3         3.0                                                  Lys            1         1.2                                                  Trp            1         --                                                   Arg            2         2.0                                          M1      Asx            2         2.1                                                  Thr            1         0.8                                                  Ser            6         5.5                                                  Glx            4         4.3                                                  Ala            4         4.6                                                  Val            2         2.3                                                  Ile            5         4.2                                                  Leu            3         3.1                                                  His            4         4.4                                                  Trp            1         --                                                   Arg            3         3.4                                          M2      Asx            5         5.1                                                  Thr            1         1.0                                                  Ser            4         3.7                                                  GLX            4         4.5                                                  Ala            2         2.2                                                  Val            2         2.3                                                  Ile            2         1.3                                                  Leu            4         3.8                                                  His            2         1.6                                                  Trp            1         --                                                   Arg            5         5.1                                                  Tyr            1         1.4                                          ______________________________________                                    

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Blalock et al., Biochem. Biophys. Res. Comm., 121:203-207, 1984.

Bost et al., Proc. Natl. Acad. Sci., 82:1372-1375, 1985.

Chang et al., "Solid-phase peptide synthesis using mild base cleavage ofN.sup.α -(-fluoroenyl)methyloxycarbonyl amino acids, exemplified by asynthesis of dihydrosomatostatin," Int. J. Pept. Protein Res., 11:246,1978.

Goodman, J. W., "Immunogenicity and Antigenic Specificity", Basic andClinical Immunology, edited by D. P. Stites and A. I. Terr., Appletonand Lange, Norwalk, Conn. pp. 101-108, 1991.

Guillet, et al., Science, 235:865-870, 1987.

Hale et al., International Immunology 1:409-415, 1989.

Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982.

Merrifield, R., J. Am. Chem. Soc. 85:2149, 1963.

Sette, et al., J. Immunol. 142:35-40, 1988.

Tam et al., Proc. Natl. Acad. Sci., 85:5409-5413, 1988.

U.S. Pat. No. 3,791,932.

U.S. Pat. No. 3,949,064.

U.S. Pat. No. 4,174,384.

U.S. Pat. No. 4,578,770.

U.S. Pat. No. 4,596,792.

U.S. Pat. No. 4,599,230.

U.S. Pat. No. 4,599,231.

U.S. Pat. No. 4,601,903.

U.S. Pat. No. 4,608,251.

U.S. Pat. No. 5,077,195.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerArgAlaValThrAlaAlaHisSerGluIle                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       HisGluAspIleIleSerLeuTrpAspGlnSerLeuArg                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerGluAlaIleIleHisValLeuHisSerArg                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaLeuAspHisSerGlyArgValArgGluThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgLeuSerTyrAsnValAspGlnMetArgAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuLysIleHisAlaGlnIleGluValSerValAla                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AlaAsnIleTyrAlaGluIleAsnSerTyrIleArg                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AspValAsnAlaTyrLeuAsnLeuArgPheHisTyr                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyLeuGlnPheHisValLysLeuTyrGlyGluAla                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlyLeuGlnPheHisValLysLeuTyrGlyGluAla                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      LeuArgPheHisTyr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlyLeuGlnPhe                                                                  __________________________________________________________________________

What is claimed is:
 1. A non-covalently interlinked multimer comprisingmonomers (A)-Ag-(a) and (B)-Ag-(b), whereinAg is an HP-6 epitope; and A,a, B, and b are independently peptide linkers of about 3 to 12 aminoacids covalently bound to the antigenic epitope; whereinpeptide linker Ahas binding affinity for peptide linker b and peptide linker a hasbinding affinity for peptide linker B due to a pattern of alternatinghydrophilic and hydrophobic amino acids or groups of amino acids.
 2. Themultimer of claim 1 wherein A and B are the same linker and a and b arethe same linker.
 3. The multimer of claim 1 wherein the pattern is ofsingly alternating hydrophilic and hydrophobic amino acid.
 4. Themultimer of claim 1 wherein the groups of amino acids include 2 or 3amino acids.
 5. The multimer of claim 1 wherein A and a are selectedfrom the group consisting of S1-L (SEQ ID NO:6) and S1-R (SEQ ID NO:7),S2-L (SEQ ID NO:8) and S2-R (SEQ ID NO:9), M1-L(SEQ ID NO:1) and M1-R(SEQ ID NO:3), M2-L (SEQ ID NO:4) and M2-R (SEQ ID NO:5), S20-L (SEQ IDNO:10) and S20-R (Gly-Leu-Gln), S22-L (SEQ ID NO:11) and S22-R (SEQ IDNO:12).
 6. The multimer of claim 1 wherein B and b are selected from thegroup consisting of S1-L (SEQ ID NO:6) and S1-R (SEQ ID NO:7), S2-L (SEQID NO:8) and S2-R (SEQ ID NO:9), M1-L(SEQ ID NO:1) and M1-R (SEQ IDNO:3), M2-L (SEQ ID NO:4) and M2-R (SEQ ID NO:5), S20-L (SEQ ID NO:10)and S20-R (Gly-Leu-Gln), S22-L (SEQ ID NO:11) and S22-R (SEQ ID NO:12).7. The multimer of claim 1 wherein the peptide linkers have spacer aminoacids.
 8. A non-covalently interlinked multimer made by a processcomprising the step of incubating a first monomer, (A)-Ag-(a), with asecond monomer, (B)-Ag-(b), under conditions facilitating peptideinteractions to form a non-covalently interlinked multimer, whereinAg isan HP-6 epitope; and A, a, B, and b are independently peptide linkers ofabout 3 to 12 amino acids; whereinpeptide linker A has non-covalentbinding affinity for peptide linker b and peptide linker a hasnon-covalent binding affinity for peptide linker B due to a pattern ofalternating hydrophilic and hydrophobic amino acids or groups of aminoacids.
 9. The multimer of claim 8 wherein the conditions facilitatedissolution of the monomers in a physiologically acceptable aqueoussolvent.
 10. A method of producing a non-covalently interlinked multimercomprising the step of incubating a first monomer, (A)-Ag-(a), with asecond monomer, (B)-Ag-(b), under conditions facilitating peptideinteractions to form a non-covalently interlinked multimer, whereinAg isan HP-6 epitope; and A, a, B, and b are independently peptide linkers ofabout 3 to 12 amino acids; whereinpeptide linker A has non-covalentbinding affinity for peptide linker b and peptide linker a has bindingaffinity for peptide linker B due to a pattern of alternatinghydrophilic and hydrophobic amino acids or groups of amino acids. 11.The method of claim 10 wherein the conditions facilitate dissolution ofthe monomers of the multimer in a physiologically acceptable aqueoussolvent.
 12. A method of producing an antibody having specific bindingaffinity for the non-covalently linked multimer of claim 1 in an animal,the method comprising the steps of obtaining an animal capable ofproducing antibody and injecting into the animal the non-covalentlyinterlinked multimer.
 13. The method of claim 12 further comprising thestep of recovering the antibody from biological fluid of the animal.